Transgenerational acclimation to acidified seawater and gene expression patterns in a sea urchin.

Transgenerational responses of susceptible calcifying organisms to progressive ocean acidification are an important issue in reducing uncertainty of future predictions. In this study, a two-generation rearing experiment was conducted using mature Mesocentrotus nudus, a major edible sea urchin that occurs along the coasts of northern Japan. Morphological observations and comprehensive gene expression analysis (RNA-seq) of resulting larvae were performed to examine transgenerational acclimation to acidified seawater. Two generations of rearing experiments showed that larvae derived from parents acclimated to acidified seawater tended to have higher survival and show less reduction in body size when exposed to acidified seawater of the same pH, suggesting that a positive carry-over effect occurred. RNA-seq analysis showed that gene expression patterns of larvae originated from both acclimated and non-acclimated parents to acidified seawater tended to be different than control condition, and the gene expression pattern of larvae originated from acclimated parents was substantially different than that of larvae of non-acclimated and control parents.

Whole transcriptome analysis of demersal fish eggs reveals complex responses to ocean deoxygenation and acidification.

Ocean acidification and deoxygenation co-occur in marine environments, causing deterioration of marine ecosystems. However, effects of compound stresses on marine organisms and their physiological coping mechanisms are largely unknown. Here, we show how high pCO2 and low dissolved oxygen (DO) cause transcriptomic changes in eggs of a demersal fish (Sillago japonica), which are fully exposed to such stresses in natural environment. Overall gene expression was affected more strongly by low DO than by high pCO2. Enrichment analysis detected significant stress responses such as glycolytic processes in response to low DO. Increased expression of a group of glycolytic genes under low DO conditions is presumably because oxygen depletion disables the electron transfer pathway, complementing ATP production in the glycolytic pathway. Contrary to expectations, apparent mitigation of gene expression changes was dominant under combined stress conditions, and may represent an innate fish adaptive trait for severe environments.

Methane diffusion affects characteristics of benthic communities in and around microbial mat-covered sediments in the northeastern Japan sea.

We investigated relationships between features of benthic macrofaunal communities and geochemical parameters in and around microbial mat-covered sediments associated with a methane seepage on Sakata Knoll in the eastern Japan Sea. A depression on top of the knoll corresponds to a gas-hydrate-bearing area with seepage of methane-rich fluid, and microbial mats cover the seafloor sediments. Sediment cores were collected at three sites for this study: one within a microbial mat, a second a few meters outside of the microbial mat, and a third from a reference site outside the gas-hydrate-bearing areas. Morphological analysis showed that the site inside the microbial mat had higher macrofaunal density and biomass compared with the other sites. 18S rRNA gene analysis showed that annelids were dominant in the surface sediment inside the microbial mat with the possible occurrence of microbial anaerobic oxidation of methane (AOM), whereas in the surface sediments outside the microbial mat and at the reference site the predominant species belonged to phylum Cercozoa. Morphological analysis also showed that the surface sediment inside the microbial mat noticeably favored annelids, with dorvilleid Ophryotrocha sp. and ampharetid Neosabellides sp. identified as major constituents. Statistical analysis showed that sulfidic sediment conditions with concentrations of H2S up to 121 µM resulting from AOM likely resulted in the predominance of annelids with tolerance to sulfide. Both the 18S rRNA genes and macrofaunal characteristics showed that benthic biodiversity among the three sites was greatest outside the microbial mat. The site outside the microbial mat may represent geochemical transition conditions, including a lower rate of upward methane gas-flow compared with the site inside the microbial mat. The high biodiversity there might result from the presence of species specifically suited to the transition zone as well as species also found in photosynthesis-based communities of the background environment.

Preliminary study on the acute effects of hydrogen sulfide on Amphipoda (Lysianassoidea; Pseudorchomene sp. and Anonyx sp.) collected from deep-sea floors in the Sea of Japan.

To study the environmental impact of the assessment technologies for the development of shallow methane hydrate zones in the Sea of Japan, deep-sea amphipods (Pseudorchomene sp. and Anonyx sp.) were collected from a depth of approximately 1000 m and were tested for H2S toxicity. At 0.57 mg L-1 H2S, all specimens of Pseudorchomene sp. were dead after 96 h, whereas all individuals survived at 0.18 mg L-1. Moreover, Anonyx sp. had a survival rate of 17 % after 96 h at 0.24 mg L-1. A similar toxicity test was conducted with the coastal amphipod Merita sp., a detritivore, and all individuals died within 24 h at 0.15 mg L-1. These results suggested that compared with coastal detritivorous amphipods, deep-sea detritivorous amphipods, which also live near biomats with sediment H2S concentrations exceeding 10 mg L-1, showed a higher tolerance to H2S.

A new hexactinellid-sponge-associated zoantharian (Porifera, Hexasterophora) from the northwestern Pacific Ocean.

Symbiotic associations between zoantharians and sponges can be divided into two groups: those that associate with Demospongiae and those that associate with Hexactinellida. Parachurabanashinseimaruae Kise, gen. nov. et sp. nov., a new genus and a new species of Hexactinellida-associated zoantharian from Japanese waters, is described. It is characterized by a combination of the following: i) its host hexactinellid sponge, ii) very flat polyps, iii) cteniform endodermal marginal muscles, and iv) characteristic mutations in three mitochondrial regions (including a unique 26-bp deletion in 16S ribosomal DNA) and three nuclear regions. Parachurabanashinseimaruae Kise, gen. nov. et sp. nov. is the third genus in the family Parazoanthidae that is reported to be associated with Hexasterophora sponges. Although specimens have so far only been collected on Takuyo-Daigo Seamount off Minami-Torishima Island in Japan, unidentified zoantharians of similar description have been reported from the waters around Australia, indicating that the species might be widespread across the Pacific.

Genetic population structures of common scavenging species near hydrothermal vents in the Okinawa Trough.

Deep-sea mining of hydrothermal deposits off the coast of Japan is currently under consideration, and environmental baseline studies of the area are required to understand possible impacts. The aim of this study is to clarify population structures of dominant benthic megafaunal species near hydrothermal vent fields in the Okinawa Trough, using a population genetics approach. We examined dominant deep-sea scavenging species including eels, several amphipods, and a decapod and performed population genetic analyses based on the mitochondrial cytochrome c oxidase subunit I region. Several sites were sampled within Okinawa Trough to examine intra-population diversity while two other locations 1400-2400 km away were chosen for inter-population comparisons. For synaphobranchid eels Simenchelys parasitica and Synaphobranchus kaupii, our results showed significant intra-population diversity but no inter-population genetic differentiation, suggesting strong genetic connectivity and/or large population sizes. In addition, single nucleotide polymorphism analysis also confirmed strong genetic connectivity for Simenchelys parasitica. Among scavenging amphipods, we detected seven putative species using molecular phylogenetic analysis. We evaluated population structures of the most abundant species of amphipods and a decapod species (Nematocarcinus lanceopes). Our results provide basic information on the genetic population structures of benthic megafaunal species near hydrothermal vent fields, which can be used to select candidate species for future connectivity analysis with high-resolution genetic markers and aid understanding of the potential population impacts of environmental disturbances.

Deep-sea amphipods around cobalt-rich ferromanganese crusts: Taxonomic diversity and selection of candidate species for connectivity analysis.

The aim of this study was to select a candidate deep-sea amphipod species suitable for connectivity analyses in areas around cobalt-rich ferromanganese crusts (CRCs). We applied DNA barcoding based on the mitochondrial protein-coding gene, cytochrome c oxidase subunit I (COI), to specimens collected from the Xufu Guyot (the JA06 Seamount) off southeastern Minami-Torishima Island in the North Pacific, where CRCs are distributed. We used baited traps to collect 37 specimens. Comparison of COI sequences with public reference databases (GenBank, BOLD) showed that almost all of the specimens belonged to the superfamily Lysianassoidea, which is known to be ubiquitous in deep-sea areas. In a molecular taxonomic analysis of these sequences, we detected 11 clades. One of these clades (group 9) composed of 18 sequences and was identified by DNA barcoding as a putative species belonging to Abyssorchomene, which has been reported from the New Hebrides Trench in the South Pacific. We considered this species to be a candidate for connectivity analysis and analyzed its genome by restriction site-associated DNA sequencing. The results showed that the genetic variation in this species is adequate for analyzing connectivity patterns in CRC areas in the future.

Microbacterium tumbae sp. nov., an actinobacterium isolated from the stone chamber of ancient tumulus.

Eight strains characterised as Gram-stain-positive, non-spore-forming and non-motile rods were isolated from samples collected from stone chambers of the Takamatsuzuka and Kitora tumuli in Asuka village, Nara Prefecture, Japan. Among them, one strain, T7528-3-6bT, was shown to form a novel lineage within the genus Microbacterium. The most closely phylogenetically related species to T7528-3-6bT was Microbacterium panaciterrae, with 97.8 % sequence similarity. The major isoprenoid quinones of T7528-3-6bT were MK-12, MK-13 and MK-11. The predominant cellular fatty acids for this isolate were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0. The diagnostic diamino acid of the peptidoglycan of this isolate was ornithine. Major polar lipids of the isolate were phosphatidylglycerol, diphosphatidylglycerol and an unknown glycolipid. The G+C content of the genomic DNA of this isolate was 70.1 mol%. On the basis of the results of physiological, biochemical and chemotaxonomic tests and molecular phylogenetic analysis, T7528-3-6bT is considered to represent a novel species of the genus Microbacterium, for which the name M. tumbae sp. nov. has been proposed. The type strain is T7528-3-6bT (=JCM 28836T=NCIMB 15039T). The results of comparisons of both phenotypic and genotypic (16S rRNA gene sequence) characteristics indicated that the remaining seven isolates were very closely related to Microbacterium shaanxiense. Although the sequence similarity between the two was 99.2 %, further detailed multifaceted comparisons are needed to determine their accurate taxonomic assignment.

Polyphasic insights into the microbiomes of the Takamatsuzuka Tumulus and Kitora Tumulus.

Microbial outbreaks and related biodeterioration problems have affected the 1300-year-old multicolor (polychrome) mural paintings of the special historic sites Takamatsuzuka Tumulus (TT) and Kitora Tumulus (KT). Those of TT are designated as a national treasure. The microbiomes of these tumuli, both located in Asuka village, Nara, Japan, are critically reviewed as the central subject of this report. Using culture-dependent methods (conventional isolation and cultivation), we conducted polyphasic studies of the these microbial communities and identified the major microbial colonizers (Fusarium spp., Trichoderma spp., Penicillium spp., dark Acremonium spp., novel Candida yeast spp., Bacillus spp., Ochrobactrum spp., Stenotrophomonas tumulicola, and a few actinobacterial genera) and noteworthy microbial members (Kendrickiella phycomyces, Cephalotrichum verrucisporum (≡Doratomyces verrucisporus), Sagenomella striatispora, Sagenomella griseoviridis, two novel Cladophialophora spp., Burgoa anomala, one novel species Prototheca tumulicola, five novel Gluconacetobacter spp., three novel Bordetella spp., and one novel genus and species Krasilnikoviella muralis) involved in the biodeterioration of mural paintings, plaster walls, and stone chamber interiors. In addition, we generated microbial community data from TT and KT samples using culture-independent methods (molecular biological methods, including PCR-DGGE, clone libraries, and pyrosequence analysis). These data are comprehensively presented, in contrast to those derived from culture-dependent methods. Furthermore, the microbial communities detected using both methods are analytically compared, and, as a result, the complementary roles of these methods and approaches are highlighted. In related contexts, knowledge of similar biodeterioration problems affecting other prehistoric cave paintings, mainly at Lascaux in France and Altamira in Spain, are referred to and commented upon. Based on substrate preferences (or ecological grouping) and mapping (plotting detection sites of isolates), we speculate on the possible origins and invasion routes whereby the major microbial colonizers invaded the TT stone chamber interior. Finally, concluding remarks, lessons, and future perspectives based on our microbiological surveys of these ancient tumuli, and similar treasures outside of Japan, are briefly presented. A list of the microbial taxa that have been identified and fully or briefly described by us as known and novel taxa for TT and KT isolates since 2008 is presented in Supplementary Materials.

Krasilnikoviella muralis gen. nov., sp. nov., a member of the family Promicromonosporaceae, isolated from the Takamatsuzuka Tumulus stone chamber interior and reclassification of Promicromonospora flava as Krasilnikoviella flava comb. nov.

A Gram-stain-positive, facultatively anaerobic actinomycete, designated strain T6220-5-2bT, was isolated from a sample taken from a mouldy spot on the surface of a mural painting (the white tiger, Byakko) inside the stone chamber of Takamatsuzuka Tumulus in Asuka village, Nara Prefecture, Japan. Based on 16S rRNA gene sequence analysis of the isolate, it was closely related to the genus Promicromonospora, but formed of a novel lineage within the family Promicromonosporaceae. The closest related species to strain T6220-5-2bT was Promicromonospora flava, with which it shared 99.1 % 16S rRNA gene sequence similarity. The isoprenoid quinone systems were menaquinones MK-9(H2), MK-9(H0) and MK-9(H4). The predominant cellular fatty acids for the isolate were anteiso-C15 : 0 and iso-C15 : 0. The peptidoglycan contained glutamic acid, aspartic acid, alanine and lysine, with the last named being the diagnostic diamino acid. The cell-wall acyl type was acetyl. The major polar lipids of the isolate were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositolmannoside, two unknown phospholipids and an unknown phosphoglycolipid. Whole-cell sugars of the isolate were galactose, glucose and ribose. The DNA G+C content of the genomic DNA was 75.2 mol%. Based on the results of phylogenetic, physiological and biochemical analyses and DNA-DNA hybridization experiments, the isolate was considered to represent a novel species of a new genus in the family Promicromonosporaceae, for which the name Krasilnikoviella muralis gen. nov., sp. nov. is proposed. The type strain of Krasilnikoviella muralis is T6220-5-2bT (=JCM 28789T=NCIMB 15040T). The reclassification of Promicromonospora flava as Krasilnikoviella flava comb. nov. is also proposed with the emended description of this species.

Genome sequence and overview of Oligoflexus tunisiensis Shr3T in the eighth class Oligoflexia of the phylum Proteobacteria.

Oligoflexus tunisiensis Shr3T is the first strain described in the newest (eighth) class Oligoflexia of the phylum Proteobacteria. This strain was isolated from the 0.2-µm filtrate of a suspension of sand gravels collected in the Sahara Desert in the Republic of Tunisia. The genome of O. tunisiensis Shr3T is 7,569,109 bp long and consists of one scaffold with a 54.3% G + C content. A total of 6,463 genes were predicted, comprising 6,406 protein-coding and 57 RNA genes. Genome sequence analysis suggested that strain Shr3T had multiple terminal oxidases for aerobic respiration and various transporters, including the resistance-nodulation-cell division-type efflux pumps. Additionally, gene sequences related to the incomplete denitrification pathway lacking the final step to reduce nitrous oxide (N2O) to nitrogen gas (N2) were found in the O. tunisiensis Shr3T genome. The results presented herein provide insight into the metabolic versatility and N2O-producing activity of Oligoflexus species.

Aurantiacicella marina gen. nov., sp. nov., a myxol-producing bacterium from surface seawater.

A Gram-stain-negative, non-motile, mesophilic, aerobic, rod-shaped bacterium, strain 2A-8T, was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. The strain produced myxol as a major carotenoid. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain fell within the family Flavobacteriaceae and was related most closely to the genus Aquimarina (91.0-94.4 % 16S rRNA gene sequence similarity to the type strains of species of this genus). The DNA G+C content was 35 mol%. The major fatty acids were iso-C15 : 0 and iso-C17 : 0 3-OH. The major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and five unidentified lipids. Menaquinone 6 was detected as the sole isoprenoid quinone. On the basis of phenotypic, genotypic and chemotaxonomic data, strain 2A-8T represents a novel genus and species, for which the name Aurantiacicella marina gen. nov., sp. nov. is proposed. The type strain of Aurantiacicella marina is 2A-8T ( = NBRC 111187T = KCTC 42676T).

Novel environmental species isolated from the plaster wall surface of mural paintings in the Takamatsuzuka tumulus: Bordetella muralis sp. nov., Bordetella tumulicola sp. nov. and Bordetella tumbae sp. nov.

Ten strains of Gram-stain-negative, non-spore-forming, non-motile coccobacilli were isolated from the plaster wall surface of 1300-year-old mural paintings inside the stone chamber of the Takamatsuzuka tumulus in Asuka village (Asuka-mura), Nara Prefecture, Japan. Based on 16S rRNA gene sequence analysis of the isolates, they belonged to the proteobacterial genus Bordetella (class Betaproteobacteria) and could be separated into three groups representing novel lineages within the genus Bordetella. Three isolates were selected, one from each group, and identified carefully using a polyphasic approach. The isolates were characterized by the presence of Q-8 as their major ubiquinone system and C16 : 0 (30.0-41.8 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 10.1-27.0 %) and C17 : 0 cyclo (10.8-23.8 %) as the predominant fatty acids. The major hydroxy fatty acids were C12 : 0 2-OH and C14 : 0 2-OH. The DNA G+C content was 59.6-60.0 mol%. DNA-DNA hybridization tests confirmed that the isolates represented three separate novel species, for which the names Bordetella muralis sp. nov. (type strain T6220-3-2bT = JCM 30931T = NCIMB 15006T), Bordetella tumulicola sp. nov. (type strain T6517-1-4bT = JCM 30935T = NCIMB 15007T) and Bordetella tumbae sp. nov. (type strain T6713-1-3bT = JCM 30934T = NCIMB 15008T) are proposed. These results support previous evidence that members of the genus Bordetella exist in the environment and may be ubiquitous in soil and/or water.

Aurantimicrobium minutum gen. nov., sp. nov., a novel ultramicrobacterium of the family Microbacteriaceae, isolated from river water.

A Gram-stain-positive, aerobic, non-motile, curved (selenoid), rod-shaped actinobacterium, designated KNCT, was isolated from the 0.2 µm-filtrate of river water in western Japan. Cells of strain KNCT were ultramicrosized (0.04-0.05 µm3). The strain grew at 15-37 °C, with no observable growth at 10 °C or 40 °C. The pH range for growth was 7-9, with weaker growth at pH 10. Growth was impeded by the presence of NaCl at concentrations greater than 1 %. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KNCT showed relatively high sequence similarity (97.2 %) to Alpinimonas psychrophila Cr8-25T in the family Microbacteriaceae. However, strain KNCT formed an independent cluster with cultured, but as-yet-unidentified, species and environmental clones on the phylogenetic tree. The major cellular fatty acids were anteiso-C15 : 0 (41.0 %), iso-C16 : 0 (21.8 %), C16 : 0 (18.0 %) and anteiso-C17 : 0 (12.9 %), and the major menaquinones were MK-11 (71.3 %) and MK-12 (13.6 %). The major polar lipids were phosphatidylglycerol and two unknown glycolipids. The cell-wall muramic acid acyl type was acetyl. The peptidoglycan was B-type, and contained 3-hydroxyglutamic acid, glutamic acid, aspartic acid, glycine, alanine and lysine, with the latter being the diagnostic diamino acid. The G+C content of the genome was unusually low for actinobacteria (52.1 mol%), compared with other genera in the family Microbacteriaceae. Based on the phenotypic characteristics and phylogenetic evidence, strain KNCT represents a novel species of a new genus within the family Microbacteriaceae, for which the name Aurantimicrobium minutum gen. nov., sp. nov. is proposed. The type strain of the type species is KNCT ( = NBRC 105389T = NCIMB 14875T).

Perspicuibacter marinus gen. nov., sp. nov., a semi-transparent bacterium isolated from surface seawater, and description of Arenicellaceae fam. nov. and Arenicellales ord. nov.

A Gram-stain-negative, non-motile, mesophilic, aerobic, rod-shaped bacterium, strain 2-9(T), was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. The strain was transparent on 1/5 strength marine broth plate but became easily visible when the plate was supplemented with pyruvate. Phylogenetic analyses based on the 16S rRNA gene sequence showed that the strain fell within the class Gammaproteobacteria and was most closely related to the genus Arenicella (92.7-93.0 % 16S rRNA gene sequence similarities to type strains of species of this genus) of an unclassified order within this class. The DNA G+C content of strain 2-9(T) was 41.7 mol%. The major fatty acids were C18 : 1ω7c (37.6 %), C16 : 1ω7c and/or iso-C15 : 0 2-OH (summed feature 3; 19.1 %), C18 : 0 (10.8 %), C16 : 0 (10.2 %) and an unidentified fatty acid with an equivalent chain-length value of 11.799 (9.5 %). The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine and three unidentified lipids. Ubiquinone-8 (Q-8) was detected as the sole isoprenoid quinone. From these taxonomic data, it is proposed that strain 2-9(T) represents a novel species of a new genus, Perspicuibacter marinus gen. nov., sp. nov. The type strain of the type species is 2-9(T) ( = NBRC 110144(T) = KCTC 42196(T)). A new family, Arenicellaceae fam. nov. (type genus Arenicella), and order, Arenicellales ord. nov., of the class Gammaproteobacteria are proposed to accommodate the novel taxon.

Flavicella marina gen. nov., sp. nov., a carotenoid-producing bacterium from surface seawater.

A Gram-stain-negative, non-motile, mesophilic, aerobic, rod-shaped or spherical bacterium, strain 2A-7(T), was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. The strain produced a pigment(s), the absorption spectrum of which closely resembled that of ß-carotene. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain fell within the family Flavobacteriaceae and clustered distantly with the type strains of species of the genus Lutibacter (up to 93.9 % similarity). The DNA G+C content was 34.1 mol%. The major fatty acids were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), iso-C17 : 0 3-OH and iso-C15 : 0 3-OH. The major polar lipids were phosphatidylethanolamine and three unidentified lipids. Menaquinone 6 was detected as the sole isoprenoid quinone. On the basis of phenotypic, genotypic and chemotaxonomic data, strain 2A-7(T) represents a novel genus and species, for which the name Flavicella marina gen. nov., sp. nov. is proposed. The type strain of Flavicella marina is 2A-7(T) ( = NBRC 110145(T) = KCTC 42197(T)).

Marinibactrum halimedae gen. nov., sp. nov., a gammaproteobacterium isolated from a marine macroalga.

Phylogenetic and taxonomic characterization was performed for a bacterium, designated strain Q-192T, isolated from the surface of the green macroalga Halimeda sp., collected from the subtropical Ishigaki Island, Japan. The isolate was a polysaccharide-producing, Gram-stain-negative, aerobic, rod-shaped, motile bacterium with a polar flagellum. The isolate was slightly halophilic, required Na+, Mg2+ and Ca2+ ions for growth, but did not require growth factors. The only isoprenoid quinone was ubiquinone-8.The major cellular fatty acids were C18 : 1ω7c, C16 : 0 and C14 : 0. The main hydroxy fatty acid was C10 : 0 3-OH. The DNA G+C content was 45.9 mol%. Phylogenetic analysis of 16S rRNA gene sequences placed the isolate in the class Gammaproteobacteria. The phylogenetically closest relatives with validly published names were Pseudomaricurvus alkylphenolicus KU41GT, Teredinibacter turnerae T7902T, Pseudoteredinibacter isoporae SW-11T and Simiduia agarivorans SA1T with sequence similarities of 94.5, 94.1, 93.7 and 93.6 %, respectively. The isolate was distinguished from members of these genera by a combination of DNA G+C content, chemotaxonomic characteristics (respiratory quinone system, fatty acid profile and polar lipid composition) and other phenotypic features. Based on phylogenetic, genotypic, chemotaxonomic and phenotypic characteristics, strain Q-192T is considered to represent a novel species of a new genus, for which the name Marinibactrum halimedae gen. nov., sp. nov. is proposed. The type strain of Marinibactrum halimedae is Q-192T ( = NBRC 110095T = NCIMB 14932T).

Amylibacter marinus gen. nov., sp. nov., isolated from surface seawater.

A Gram-stain-negative, non-motile, mesophilic, aerobic, rod-shaped bacterium, designated strain 2-3(T), was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. This strain grew well with starch. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain fell within the family Rhodobacteraceae and that the strain was related most closely to the genus Pacificibacter (94.0 % sequence similarity to the type strain). The DNA G+C content was 52.4 mol%. The major fatty acids were C18 : 1ω7c, C14 : 0 and C16 : 0. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, one unidentified lipid, one unidentified aminolipid and one unidentified phospholipid. The major isoprenoid quinone was Q-10. Strain 2-3(T) did not grow at 4 or 35 °C, while the type strain of the type species of the genus Pacificibacter grows at both temperatures. From the taxonomic data obtained in this study, it is proposed that strain 2-3(T) be placed into a novel genus and species named Amylibacter marinus gen. nov., sp. nov. in the family Rhodobacteraceae. The type strain of Amylibacter marinus is 2-3(T) ( = NBRC 110140(T) = LMG 28364(T)).

Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing.

For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification of 16S rDNA is a critical step. To obtain accurate microbial composition data, PCR amplification must be free of bias; however, amplifying all 16S rDNA species with equal efficiency from a sample containing a large variety of microorganisms remains challenging. Here, we designed a universal primer based on the V3-V4 hypervariable region of prokaryotic 16S rDNA for the simultaneous detection of Bacteria and Archaea in fecal samples from crossbred pigs (Landrace × Large white × Duroc) using an Illumina MiSeq next-generation sequencer. In-silico analysis showed that the newly designed universal prokaryotic primers matched approximately 98.0% of Bacteria and 94.6% of Archaea rRNA gene sequences in the Ribosomal Database Project database. For each sequencing reaction performed with the prokaryotic universal primer, an average of 69,330 (± 20,482) reads were obtained, of which archaeal rRNA genes comprised approximately 1.2% to 3.2% of all prokaryotic reads. In addition, the detection frequency of Bacteria belonging to the phylum Verrucomicrobia, including members of the classes Verrucomicrobiae and Opitutae, was higher in the NGS analysis using the prokaryotic universal primer than that performed with the bacterial universal primer. Importantly, this new prokaryotic universal primer set had markedly lower bias than that of most previously designed universal primers. Our findings demonstrate that the prokaryotic universal primer set designed in the present study will permit the simultaneous detection of Bacteria and Archaea, and will therefore allow for a more comprehensive understanding of microbial community structures in environmental samples.

Oligoflexus tunisiensis gen. nov., sp. nov., a Gram-negative, aerobic, filamentous bacterium of a novel proteobacterial lineage, and description of Oligoflexaceae fam. nov., Oligoflexales ord. nov. and Oligoflexia classis nov.

A phylogenetically novel proteobacterium, strain Shr3(T), was isolated from sand gravels collected from the eastern margin of the Sahara Desert. The isolation strategy targeted bacteria filterable through 0.2-µm-pore-size filters. Strain Shr3(T) was determined to be a Gram-negative, aerobic, non-motile, filamentous bacterium. Oxidase and catalase reactions were positive. Strain Shr3(T) showed growth on R2A medium, but poor or no growth on nutrient agar, trypticase soy agar and standard method agar. The major isoprenoid quinone was menaquinone-7. The dominant cellular fatty acids detected were C16:1ω5c and C16:0, and the primary hydroxy acid present was C12:0 3-OH. The DNA G+C content was 54.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Shr3(T) was affiliated with an uncultivated lineage of the phylum Proteobacteria; the nearest known type strain, with 83% sequence similarity, was Desulfomicrobium orale DSM 12838(T) in the class Deltaproteobacteria. The isolate and closely related environmental clones formed a novel class-level clade in the phylum Proteobacteria with high bootstrap support (96-99%). Based on these results, the novel class Oligoflexia classis nov. in the phylum Proteobacteria and the novel genus and species Oligoflexus tunisiensis gen. nov., sp. nov. are proposed for strain Shr3(T), the first cultivated representative of the Oligoflexia. The type strain of Oligoflexus tunisiensis is Shr3(T) ( = JCM 16864(T) = NCIMB 14846(T)). We also propose the subordinate taxa Oligoflexales ord. nov. and Oligoflexaceae fam. nov. in the class Oligoflexia.