Helicovermis profundi gen. nov., sp. nov., a novel mesophilic, asporogenous bacterium within the Clostridia isolated from a deep-sea hydrothermal vent chimney.

A novel mesophilic bacterial strain, designated S502T, was isolated from a deep-sea hydrothermal vent at Suiyo Seamount, Japan. Cells were Gram-positive, asporogenous, motile, and curved rods, measuring 1.6-5.6 µm in length. The strain was an obligate anaerobe that grew fermentatively on complex substrates such as yeast extract and Bacto peptone. Elemental sulfur stimulated the growth of the strain, and was reduced to hydrogen sulfide. The strain grew within a temperature range of 10-23 °C (optimum at 20 °C), pH range of 4.8-8.3 (optimum at 7.4), and a NaCl concentration range of 1.0-4.0% (w/v) (optimum at 3.0%, w/v). Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolate was a member of the class Clostridia, with Fusibacter paucivorans strain SEBR 4211T (91.1% sequence identity) being its closest relative. The total size of the genome of the strain was 3.12 Mbp, and a G + C content was 28.2 mol%. The highest values for average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) value of strain S502T with relatives were 67.5% (with Marinisporobacter balticus strain 59.4MT), 51.5% (with M. balticus strain 59.4MT), and 40.9% (with Alkaliphilus serpentinus strain LacTT), respectively. Based on a combination of phylogenetic, genomic, and phenotypic characteristics, we propose strain S502T to represent a novel genus and species, Helicovermis profundi gen. nov., sp. nov., with the type strain S502T (= DSM 112048T = JCM 39167T).

Rapid Freezing and Cryo-SEM-EDS Imaging of Foraminifera (Unicellular Eukaryotes) Using a Conductive Viscous Cryogenic Glue.

Spatial distribution of water-soluble molecules and ions in living organisms is still challenging to assess. Energy-dispersive X-ray spectroscopy (EDS) via cryogenic scanning electron microscopy (cryo-SEM) is one of the promising methods to study them without loss of dissolved contents. High-resolution cryo-SEM-EDS has challenges in sample preparation, including cross-section exposure and sample drift/charging due to insulative surrounding water. The former becomes problematic for large and inseparable organisms, such as benthic foraminifera, a unicellular eukaryote playing significant roles in marine ecosystems, which often exceed the size limit for the most reliable high-pressure freezing. Here we show graphite oxide dispersed in sucrose solution as a good glue to freeze, expose cross-section by cryo-ultramicrotome, and analyze elemental distribution owing to the glue's high viscosity, adhesion force, and electron conductivity. To demonstrate the effectiveness and applicability of the glue for cryo-SEM-EDS, deep-sea foraminifer Uvigerina akitaensis was sampled during a cruise and plunge frozen directly on the research vessel, where the liquid nitrogen supply is limited. The microstructures were preserved as faithfully in cryo-SEM images as those with the conventional resin-substituted transmission electron micrograph. We found elements colocalized within the cytoplasm originating from water-soluble compounds that can be lost with conventional dehydrative fixation.

Haliovirga abyssi gen. nov., sp. nov., a mesophilic fermentative bacterium isolated from the Iheya North hydrothermal field, and proposal of Haliovirgaceae fam. nov.

A novel mesophilic, obligately anaerobic, facultatively sulphur-reducing bacterium, designated strain IC12T, was isolated from a deep-sea hydrothermal field in the Mid-Okinawa Trough, Japan. The cells were Gram-negative, motile, short rods with a single polar flagellum. The ranges and optima of the growth temperature, NaCl concentration and pH of strain IC12T were 15-40 °C (optimum, 30-35 °C), 10-60 g l-1 (optimum, 20-30 g l-1) and pH 4.9-6.7 (optimum, pH 5.8), respectively. Yeast extract was utilized as a sole carbon and energy source for fermentative growth. Major fatty acids of strain IC12T were C14 : 0, C16 : 0 and C16 : 1 ω7. Results of phylogenetic analysis based on 16S rRNA gene sequences indicated that strain IC12T was affiliated to the phylum Fusobacteriota and was most closely related to Ilyobacter insuetus VenChi2T (86.5 % sequence similarity). Strain IC12T contained a chromosome of 2.43 Mbp and a large plasmid of 0.30 Mbp. The G+C content of the genomic DNA was 26.4 mol%. The maximum values for average nucleotide identity and in silico DNA-DNA hybridization between strain IC12T and related strains of the phylum Fusobacteriota were 71.4 and 26.4 %, respectively. Phylogenomic, physiological and chemotaxonomic analyses indicate that strain IC12T represents a novel genus and species within the phylum Fusobacteriota, for which the name Haliovirga abyssi gen. nov., sp. nov. is proposed, with strain IC12T (= DSM 112164T=JCM 39166T) as the type strain. We also propose the family Haliovirgaceae fam. nov. to accommodate this novel genus.

Podomonas kaiyoae n. sp., a novel apusomonad growing axenically.

Apusomonadida (apusomonads) is a group of heterotrophic biflagellates that feed on bacteria and small protists. Their diversity is not fully understood, and several major lineages remain to be identified in natural environments. Here, we report Podomonas kaiyoae n. sp., which was isolated from deep-sea sediment and can be maintained as an axenic culture. While P. kaiyoae branched within one of the major unidentified lineages, the combination of the morphological characteristics is generally similar to that of Podomonas species, but can be distinguished from that of other Podomonas species based on the cell sizes.

Pyrofollis japonicus gen. nov. sp. nov., a novel member of the family Pyrodictiaceae isolated from the Iheya North hydrothermal field.

A novel hyperthermophilic, heterotrophic archaeon, strain YC29T, was isolated from a deep-sea hydrothermal vent in the Mid-Okinawa Trough, Japan. Cells of strain YC29T were non-motile, irregular cocci with diameters of 1.2-3.0 µm. The strain was an obligatory fermentative anaerobe capable of growth on complex proteinaceous substrates. Growth was observed between 85 and 100 °C (optimum 90-95 °C), pH 4.9-6.4 (optimum 5.1), and in the presence of 1.4-4.0% (w/v) NaCl (optimum 3.0%). Inorganic carbon was required as a carbon source. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolate was a member of the family Pyrodictiaceae. The genome size was 2.02 Mbp with a G+C content of 49.4%. The maximum values for average nucleotide identity (ANI), average amino acid identity (AAI), and in silico DNA-DNA hybridization (dDDH) value of strain YC29T with relatives were 67.9% (with Pyrodictium abyssi strain AV2T), 61.1% (with Pyrodictium occultum strain PL-19T), and 33.8% (with Pyrolobus fumarii strain 1AT), respectively. Based on the phylogenetic, genomic, and phenotypic characteristics, we propose that strain YC29T represents a novel genus and species, Pyrofollis japonicus gen. nov., sp. (type strain YC29T = DSM 113394T = JCM 39171T).

Multispecies Populations of Methanotrophic Methyloprofundus and Cultivation of a Likely Dominant Species from the Iheya North Deep-Sea Hydrothermal Field.

The Methyloprofundus clade is represented by uncultivated methanotrophic bacterial endosymbionts of deep-sea bathymodiolin mussels, but only a single free-living species has been cultivated to date. This study reveals the existence of free-living Methyloprofundus variants in the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. A clade-targeted amplicon analysis of the particulate methane monooxygenase gene (pmoA) detected 647 amplicon sequence variants (ASVs) of the Methyloprofundus clade in microbial communities newly formed in in situ colonization systems. Such systems were deployed at colonies of bathymodiolin mussels and a galatheoid crab in diffuse-flow areas. These ASVs were classified into 161 species-like groups. The proportion of the species-like groups representing endosymbionts of mussels was unexpectedly low. A methanotrophic bacterium designated INp10, a likely dominant species in the Methyloprofundus population in this field, was enriched in a biofilm formed in a methane-fed cultivation system operated at 10°C. Genomic characterization with the gene transcription data set of INp10 from the biofilm suggested traits advantageous to niche competition in environments, such as mobility, chemotaxis, biofilm formation, offensive and defensive systems, and hypoxia tolerance. The notable metabolic traits that INp10 shares with some Methyloprofundus members are the use of lanthanide-dependent XoxF as the sole methanol dehydrogenase due to the absence of the canonical MxaFI, the glycolytic pathway using fructose-6-phosphate aldolase instead of fructose-1,6-bisphosphate aldolase, and the potential to perform partial denitrification from nitrate under oxygen-limited conditions. These findings help us better understand the ecological strategies of this possibly widespread marine-specific methanotrophic clade. IMPORTANCE The Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough is characterized by abundant methane derived from organic-rich sediments and diverse chemosynthetic animal species, including those harboring methanotrophic bacterial symbionts, such as bathymodiolin mussels Bathymodiolus japonicus and "Bathymodiolus" platifrons and a galatheoid crab, Shinkaia crosnieri. Symbiotic methanotrophs have attracted significant attention, and yet free-living methanotrophs in this environment have not been studied in detail. We focused on the free-living Methyloprofundus spp. that thrive in this hydrothermal field and identified an unexpectedly large number of species-like groups in this clade. Moreover, we enriched and characterized a methanotroph whose genome sequence indicated that it corresponds to a new species in the genus Methyloprofundus. This species might be a dominant member of the indigenous Methyloprofundus population. New information on free-living Methyloprofundus populations suggests that the hydrothermal field is a promising locale at which to investigate the adaptive capacity and associated genetic diversity of Methyloprofundus spp.

Desulfomarina profundi gen. nov., sp. nov., a novel mesophilic, hydrogen-oxidizing, sulphate-reducing chemolithoautotroph isolated from a deep-sea hydrothermal vent chimney.

A novel mesophilic, strictly anaerobic, chemolithoautotrophic sulphate-reducing bacterium, designated strain KT2T, was isolated from a deep-sea hydrothermal vent chimney at the Suiyo Seamount in the Izu-Bonin Arc. Strain KT2T grew at 25-40 °C (optimum 35 °C) and pH 5.5-7.0 (optimum 6.6) in the presence of 25-45 g l-1 NaCl (optimum 30 g l-1). Growth occurred with molecular hydrogen as the electron donor and sulphate, thiosulphate, and sulphite as the electron acceptors. The isolate utilized CO2 as the sole carbon source for chemolithoautotrophic growth on H2. Glycerol, succinate, fumarate, malate, glutamate, or casamino acids could serve as an alternative electron donor in the presence of CO2. Malate, citrate, glutamate, and casamino acids were used as fermentative substrates for weak growth. The G+C content of genomic DNA was 46.1 %. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain KT2T is a member of the family Desulfobulbaceae, showing a sequence similarity of 94.3 % with Desulforhopalus singaporensis. Phylogenomic analysis based on concatenated 156 single-copy marker genes confirmed the same topology as the 16S rRNA gene phylogeny. The ANI and AAI values between strain KT2T and related genera of the family Desulfobulbaceae were 65.6-68.6 % and 53.1-62.9 %. Based on the genomic, molecular, and physiological characteristics, strain KT2T represents a novel genus and species within the family Desulfobulbaceae, for which the name Desulfomarina profundi gen. nov., sp. nov. is proposed, with KT2T (=JCM 34118T = DSM 111364T) as the type strain.

Abundant Chitinous Structures in Chilostomella (Foraminifera, Rhizaria) and Their Potential Functions.

Benthic foraminifera, members of Rhizaria, inhabit a broad range of marine environments and are particularly common in hypoxic sediments. The biology of benthic foraminifera is key to understanding benthic ecosystems and relevant biogeochemical cycles, especially in hypoxic environments. Chilostomella is a foraminiferal genus commonly found in hypoxic deep-sea sediments and has poorly understood ecological characteristics. For example, the carbon isotopic compositions of their lipids are substantially different from other co-occurring genera, probably reflecting unique features of its metabolism. Here, we investigated the cytoplasmic and ultrastructural features of Chilostomella ovoidea from bathyal sediments of Sagami Bay, Japan, based on serial semi-thin sections examined using an optical microscope followed by a three-dimensional reconstruction, combined with TEM observations of ultra-thin sections. Observations by TEM revealed the presence of abundant electron-dense structures dividing the cytoplasm. Based on histochemical staining, these structures are shown to be composed of chitin. Our 3D reconstruction revealed chitinous structures in the final seven chambers. These exhibited a plate-like morphology in the final chambers but became rolled up in earlier chambers (toward the proloculus). These chitinous, plate-like structures may function to partition the cytoplasm in a chamber to increase the surface/volume ratio and/or act as a reactive site for some metabolic functions.

Symbiont Transmission onto the Cell Surface of Early Oocytes in the Deep-Sea Clam Phreagena okutanii.

Symbiotic associations with beneficial microorganisms endow a variety of host animals with adaptability to the environment. Stable transmission of symbionts across host generations is a key event in the maintenance of symbiotic associations through evolutionary time. However, our understanding of the mechanisms of symbiont transmission remains fragmentary. The deep-sea clam Phreagena okutanii harbors chemoautotrophic intracellular symbiotic bacteria in gill epithelial cells, and depends on these symbionts for nutrition. In this study, we focused on the association of these maternally transmitted symbionts with ovarian germ cells in juvenile female clams. First, we established a sex identification method for small P. okutanii individuals, and morphologically classified female germ cells observed in the ovary. Then, we investigated the association of the endosymbiotic bacteria with germ cells. We found that the symbionts were localized on the outer surface of the cell membrane of primary oocytes and not within the cluster of oogonia. Based on our findings, we discuss the processes and mechanisms of symbiont vertical transmission in P. okutanii.

Identification of cells expressing two peptidoglycan recognition proteins in the gill of the vent mussel, Bathymodiolus septemdierum.

In symbiotic systems in which symbionts are transmitted horizontally, hosts must accept symbionts from the environment while defending themselves against invading pathogenic microorganisms. How they distinguish pathogens from symbionts and how the latter evade host immune defences are not clearly understood. Recognition of foreign materials is one of the most critical steps in stimulating immune responses, and pattern recognition receptors (PRRs) play vital roles in this process. In this study, we focused on a group of highly conserved PRRs, peptidoglycan recognition proteins (PGRPs), in the deep-sea mussel, Bathymodiolus septemdierum, which harbours chemosynthetic bacteria in their gill epithelial cells. We isolated B. septemdierum PGRP genes BsPGRP-S and BsPGRP-L, which encode a short- and a long-type PGRP, respectively. The short-type PGRP has a signal peptide and was expressed in the asymbiotic goblet mucous cells in the gill epithelium, whereas the long-type PGRP was predicted to include a transmembrane domain and was expressed in gill bacteriocytes. Based on these findings, we hypothesize that the secreted and transmembrane PGRPs are engaged in host defence against pathogenic bacteria and/or in the regulation of symbiosis via different cellular localizations and mechanisms.

Morphological and functional characterization of hemocytes from two deep-sea vesicomyid clams Phreagena okutanii and Abyssogena phaseoliformis.

Deep-sea vesicomyid clams harboring intracellular symbiotic sulfur-oxidizing bacteria are often dominant in chemosynthetic animal communities. Although they are known to have erythrocytes, little is known about other hemocytes. To investigate the types and roles of various hemocytes in vesicomyid clams, we performed morphological, histochemical and functional characterization of the hemocytes in two species, Phreagena okutanii, collected from 873 to 978 m depth, and Abyssogena phaseoliformis, from 5199 to 5355 m. Both were found to have three types of hemocytes: erythrocytes (ERCs), eosinophilic granulocytes (EGs), and basophilic granulocytes (BGs). The ERCs contain hemoglobin in the cytoplasm, with basophilic vacuoles containing acid polysaccharide, neutral lipids, and peroxidase. The EGs were found to contain acid polysaccharides and eosinophilic granules containing lysosomal enzymes, acid and alkaline phosphatases, chloroacetate esterase, and peroxidase. Although BGs had some basophilic granules with alkaline phosphatase, they lacked acid phosphatase and acid polysaccharides. The EGs and BGs were shown to have phagocytic ability, while the ERCs exhibited no phagocytosis. The EGs showed higher phagocytic activity as well as a higher phagosome-lysosome fusion rate than BGs. The hemocytes of the two vesicomyid species differed in the intracellular structures. In A. phaseoliformis, ERCs additionally contained neutral polysaccharides in vacuoles and had vesicles with acinus-like acidic mucus in the cytoplasm, neither of which were observed in P. okutanii. The eosinophilic granules in the EGs had heteromorphically-elongated shapes containing homogeneously electron-dense material in P. okutanii, but were more spherical and composed of fibrous structures in A. phaseoliformis. The difference in hemocytes between the two clams seems to be reflective of phylogenetically differentiated lineages adapting to differing conditions in their respective deep-sea environments, such as dissolved oxygen, hydrogen sulfide concentration, and hydrostatic pressure. In the view of phylogeny of veneroida clams including two vesicomyids, their hemocytes appear to be categorizable into three basic types, with the first containing ERCs and agranulocytes, the second including EGs, and the third comprised of BGs, small eosinophilic granulocytes, and other granulocytes. The present data showed no phagocytic activity of ERCs and a lack of agranulocytes in both vesicomyid species, and when combined with previous reports that other veneroid clams show low or no phagocytic activity, this suggests that ERCs have become evolutionarily differentiated from agranulocytes in the ancestral vesicomyid clam.

Involvement of flocculin in negative potential-applied ITO electrode adhesion of yeast cells.

The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between -0.2 and -0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications.

Aneurinibacillus tyrosinisolvens sp. nov., a tyrosine-dissolving bacterium isolated from organics- and methane-rich seafloor sediment.

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).

Phylogeny and Reclassification of Hemistasia phaeocysticola (Scherffel) Elbrächter & Schnepf, 1996.

Hemistasia phaeocysticola is a marine flagellate that preys on diatoms and dinoflagellates among others. Although its morphology and ultrastructure were previously observed and characterized, its phylogenetic position has not been analyzed using molecular sequence data. This flagellate was classified as a kinetoplastid on the basis of the presence of a kinetoplast in the mitochondrion. However, several morphological characteristics similar to those of diplonemids, a sister group of kinetoplastids, have also been noted. Herein, we report that H. phaeocysticola branches within the diplonemid clade in the phylogenetic tree reconstructed by analyzing 18S rRNA gene sequences. Its systematic placement based on this finding is also discussed.

Phagocytic activities of hemocytes from the deep-sea symbiotic mussels Bathymodiolus japonicus, B. platifrons, and B. septemdierum.

Deep-sea mytilid mussels harbor symbiotic bacteria in their gill epithelial cells that are horizontally or environmentally transmitted to the next generation of hosts. To understand the immune defense system in deep-sea symbiotic mussels, we examined the hemocyte populations of the symbiotic Bathymodiolus mussel species Bathymodiolus japonicus, Bathymodiolus platifrons, and Bathymodiolus septemdierum, and characterized three types of hemocytes: agranulocytes (AGs), basophilic granulocytes (BGs), and eosinophilic granulocytes (EGs). Of these, the EG cells were the largest (diameter, 8.4-10.0 µm) and had eosinophilic cytoplasm with numerous eosinophilic granules (diameter, 0.8-1.2 µm). Meanwhile, the BGs were of medium size (diameter, 6.7-8.0 µm) and contained small basophilic granules (diameter, 0.3-0.4 µm) in basophilic cytoplasm, and the AGs, the smallest of the hemocytes (diameter, 4.8-6.0 µm), had basophilic cytoplasm lacking granules. A lectin binding assay revealed that concanavalin A bound to all three hemocyte types, while wheat germ agglutinin bound exclusively to EGs and BGs. The total hemocyte population densities within the hemolymph of all three Bathymodiolus mussel species were similar (8.4-13.3 × 10(5) cells/mL), and the percentages of circulating AGs, BGs, and EGs in the hemolymph of these organisms were 44.7-48.5%, 14.3-17.6%, and 34.3-41.0%, respectively. To analyze the functional differences between these hemocytes, the phagocytic activity and post-phagocytic phagosome-lysosome fusion events were analyzed in each cell type using a fluorescent Alexa Fluor(®) 488-conjugated Escherichia coli bioparticle and a LysoTracker(®) lysosomal marker, respectively. While the AGs exhibited no phagocytic activity, both types of granulocytes were phagocytic. Of the three hemocyte types, the EGs exhibited the highest level of phagocytic activity as well as rapid phagosome-lysosome fusion, which occurred within 2 h of incubation. Meanwhile, the BGs showed lower phagocytic activity and lower rates of phagosome-lysosome fusion than the EGs. These findings indicate that the two types of granulocyte play distinct roles in the defense system.

Brevundimonas denitrificans sp. nov., a denitrifying bacterium isolated from deep subseafloor sediment.

A novel Gram-stain-negative, aerobic, heterotrophic, stalked and capsulated bacterium with potential denitrification ability, designated strain TAR-002(T), 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-002(T) belongs to the genus Brevundimonas of the class Alphaproteobacteria. Levels of similarity between the 16S rRNA gene sequence of strain TAR-002(T) 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-002(T) and its phylogenetic neighbours, Brevundimonas lenta DS-18(T), B. basaltis J22(T), Brevundimonas subvibrioides ATCC 15264(T) and Brevundimonas alba DSM 4736(T), mean hybridization levels were 6.4-27.7%. The G+C content of strain TAR-002(T) was 70.3 mol%. Q-10 was the major respiratory isoprenoid quinone. The major fatty acids were C(18:1)ω7c and C(16:0), 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-002(T) with the genus Brevundimonas. On the basis of biological characteristics and 16S rRNA gene sequence comparisons, strain TAR-002(T) 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-002(T) ( =NBRC 110107(T) =CECT 8537(T)).

Thalassospira alkalitolerans sp. nov. and Thalassospira mesophila sp. nov., isolated from a decaying bamboo sunken in the marine environment, and emended description of the genus Thalassospira.

Two marine bacteria, designated strains MBE#61(T) and MBE#74(T), 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#61(T) was spiral, whereas MBE#74(T) was rod-shaped. The temperature, pH and salt concentration ranges for growth of strain MBE#61(T) 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#74(T) 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#61(T) and those of the type strains of species of the genus Thalassospira was 97.5-99.0 %, and that of strain MBE#74(T) was 96.9-98.6 %; these two isolates were most closely related to Thalassospira lucentensis QMT2(T). However, the DNA-DNA hybridization values between T. lucentensis QMT2(T) and strain MBE#61(T) or MBE#74(T) were only 16.0 % and 7.1 %, respectively. The DNA G+C content of strain MBE#61(T) was 54.4 mol%, and that of strain MBE#74(T) was 55.9 mol%. The predominant isoprenoid quinone of the two strains was Q-10 (MBE#61(T), 97.3 %; MBE#74(T), 93.5 %). The major cellular fatty acids of strain MBE#61(T) 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#74(T) were C16 : 0 (26.2 %), C17 : 0 cyclo (19.9 %) and C18 : 1ω7c (12.1 %). On the basis of these results, strain MBE#61(T) and strain MBE#74(T) 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#61(T) ( = JCM 18968(T) = CECT 8273(T)) and MBE#74(T) ( = JCM 18969(T) = CECT 8274(T)), respectively. An emended description of the genus Thalassospira is also proposed.

Loktanella cinnabarina sp. nov., isolated from a deep subseafloor sediment, and emended description of the genus Loktanella.

A Gram-stain-negative, aerobic, heterotrophic and salt-tolerant bacterium, designated strain LL-001(T), 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-001(T) belonged to the genus Loktanella within the family Rhodobacteraceae of the class Alphaproteobacteria. 16S rRNA gene sequence similarity between strain LL-001(T) and members of the genus Loktanella was 94.5-98.5 %; the highest sequence similarity was with Loktanella hongkongensis UST950701-009P(T). DNA-DNA relatedness between strain LL-001(T) and L. hongkongensis UST950701-009P(T) was 41.5-43.6 %. The DNA G+C content of strain LL-001(T) was 69.3 mol%. On the basis of biochemical features and 16S rRNA gene sequence comparison, strain LL-001(T) 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-001(T) ( = JCM 18161(T) = CECT 8072(T)). The description of the genus Loktanella is also emended.

Brevundimonas abyssalis sp. nov., a dimorphic prosthecate bacterium isolated from deep-subsea floor sediment.

A novel Gram-negative, aerobic, psychrotolerant, alkali-tolerant, heterotrophic and dimorphic prosthecate bacterium, designated strain TAR-001(T), 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-001(T) 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-001(T) 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-001(T) 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-001(T) is more closely to the genus Brevundimonas than to the genus Caulobacter. The mean DNA-DNA hybridization levels between strain TAR-001(T) 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-001(T) 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-001(T) (=JCM 18150(T)=CECT 8073(T)).

Polycladomyces abyssicola gen. nov., sp. nov., a thermophilic filamentous bacterium isolated from hemipelagic sediment.

A novel filamentous bacterium, designated strain JIR-001(T), 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-001(T) belongs to the family Thermoactinomycetaceae within the class Bacilli. Similarity levels between the 16S rRNA gene sequence of strain JIR-001(T) and those of the type strains of Thermoactinomycetaceae species were 85.5-93.5%; highest sequence similarity was with Melghirimyces algeriensis NariEX(T). In the DNA-DNA hybridization assays between strain JIR-001(T) and its phylogenetic neighbours the mean hybridization levels with Melghirimyces algeriensis NariEX(T), Planifilum fimeticola H0165(T), Planifilum fulgidum 500275(T) and Planifilum yunnanense LA5(T) 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-001(T) 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-001(T) 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-001(T) (=JCM 18147(T)=CECT 8074(T)).