Kaoru Yamaguchi

Caldisericum exile gen. nov., sp. nov., an anaerobic, thermophilic, filamentous bacterium of a novel bacterial phylum, Caldiserica phyl. nov., originally called the candidate phylum OP5, and description of Caldisericaceae fam. nov., Caldisericales ord. nov. and Caldisericia classis nov.

An anaerobic, thermophilic, thiosulfate-reducing bacterium, strain AZM16c01(T), isolated from a hot spring in Japan [Mori, K., Sunamura, M., Yanagawa, K., Ishibashi, J., Miyoshi, Y., Iino, T., Suzuki, K. & Urabe, T. (2008). Appl Environ Microbiol 74, 6223-6229] was characterized in detail. The 16S rRNA gene sequence analysis had revealed that strain AZM16c01(T) was the first cultivated representative of the candidate phylum OP5. The cells were multicellular filaments with a single polar flagellum. The strain contained iso-C(17 : 0) as the major fatty acid and menaquinone-8(H(6)), menaquinone-8(H(8)) and menaquinone-8(H(10)) as the respiratory quinones. The G+C content of the genomic DNA of strain AZM16c01(T) was 34.6 mol%. Optimum growth was obtained at 65 degrees C, pH 6.5 and in the absence of NaCl, with a doubling time of 10.6 h. On the basis of the results of phylogenetic analysis based on the 16S rRNA gene sequence and the characterization of the strain in this study, we propose the name Caldisericum exile gen. nov., sp. nov. for strain AZM16c01(T) (=NBRC 104410(T)=DSM 21853(T)). In addition, we propose the new phylum name Caldiserica phyl. nov. for the candidate phylum OP5 represented by C. exile gen. nov., sp. nov., and Caldisericaceae fam. nov., Caldisericales ord. nov. and Caldisericia classis nov.

Aceticlastic and NaCl-Requiring Methanogen “Methanosaeta pelagica” sp. nov., Isolated from Marine Tidal Flat Sediment

ABSTRACT Among methanogens, only 2 genera, Methanosaeta and Methanosarcina, are known to contribute to methanogenesis from acetate, and Methanosaeta is a specialist that uses acetate specifically. However, Methanosaeta strains so far have mainly been isolated from anaerobic digesters, despite the fact that it is widespread, not only in anaerobic methanogenic reactors and freshwater environments, but also in marine environments, based upon extensive 16S rRNA gene-cloning analyses. In this study, we isolated an aceticlastic methanogen, designated strain 03d30qT, from a tidal flat sediment. Phylogenetic analyses based on 16S rRNA and mcrA genes revealed that the isolate belongs to the genus Methanosaeta. Unlike the other known Methanosaeta species, this isolate grows at Na+ concentrations of 0.20 to 0.80 M, with an optimum concentration of 0.28 M. Quantitative estimation using real-time PCR detected the 16S rRNA gene of the genus Methanosaeta in the marine sediment, and relative abundance ranged from 3.9% to 11.8% of the total archaeal 16S rRNA genes. In addition, the number of Methanosaeta organisms increased with increasing depth and was much higher than that of Methanosarcina organisms, suggesting that aceticlastic methanogens contribute to acetate metabolism to a greater extent than previously thought in marine environments, where sulfate-reducing acetate oxidation prevails. This is the first report on marine Methanosaeta species, and based on phylogenetic and characteristic studies, the name “Methanosaeta pelagica” sp. nov. is proposed for this novel species, with type strain 03d30q.

Examination of electron stains as a substitute for uranyl acetate for the ultrathin sections of bacterial cells

Electron staining reagents were examined to find a possible substitute for uranyl acetate (UA) in electron microscopy of bacterial ultrathin sections. Four kinds of stains, platinum blue (Pt-blue), oolong tea extract (OTE), potassium permanganate (KMnO(4)) and phosphotungstic acid (PTA), were examined in comparison with UA either with or without post-staining with lead citrate (Pb). Electron microscopy was performed on sections from Spurr-embedded cells of a Gram-positive bacterium, Bacillus cereus NBRC 13597, and a Gram-negative bacterium, Escherichia coli NBRC 3301. Both Pt-blue and OTE showed staining similar to each other and to that of double staining with UA and Pb in B. cereus, while in E. coli the cytoplasmic membrane appeared less dense when compared with UA and Pb. KMnO(4) stained excessively to some extent, but showed images of the best contrast in the cytoplasmic membrane comparable with UA and Pb among the four reagents. PTA could stain the peptidoglycan layer but gave images of low quality for both bacteria. This study demonstrated that none of the reagents examined showed staining results of the same quality or better than the conventional method with UA and Pb. However, stains of Pt-blue, OTE and KMnO(4) could possibly be an alternative candidate for the UA according to the structure in question.

Trichoderma matsushimae and T. aeroaquaticum: two aero-aquatic species with Pseudaegerita-like propagules

Four isolates tentatively identified as Pseudaegerita matsushimae on the basis of the morphology of bulbil-like propagules were collected from substrates submerged in water in Thailand and Japan. In culture studies the two Thai isolates were found to produce phialoconidia on conidiogenous cells and phialoconidiophores whose morphology was similar to that of Trichoderma. Phylogenetic analysis based on D1/D2 regions of LSU rDNA sequences showed that the four isolates were nested in Hypocrea/Trichoderma (Hypocreales) while P. corticalis, the type species of Pseudaegerita, belongs to Hyaloscypha (Helotiales). Preliminary analysis by ISTH Web tools based on 5.8S-ITS rDNA and phylogenetic analysis based on rpb2 and tef1-int4 genes showed that the isolates have specific sequences of Trichoderma (anchors 1–5) and belong to the Hamatum clade but they grouped apart from any known species of Trichoderma. The sequences of the tef1-int4 gene, which were amplified from the authentic specimen of P. matsushimae (IMI 266915), also showed that it belongs to the Hamatum clade closely clustering with T. yunnanense but separate from our four isolates. The morphology of P. matsushimae (IMI 266915), especially the sizes of phialides and phialoconidia, were different from T. yunnanense. Thus, we conclude that IMI 266915 and our isolates are to be assigned to two different species in the Hamatum clade of Trichoderma, although both species have similar morphology of bulbils and phialoconidia. Morphology and molecular data revealed that P. matsushimae should be assigned to the genus Trichoderma as T. matsushimae and the Thai and Japanese isolates are placed in T. aeroaquaticum sp. nov. This finding supports the interpretation that aero-aquatic fungi have evolved from terrestrial fungi. We assume that these fungi probably were derived from typically soil-inhabiting species of Trichoderma; an adaptation to aquatic environments is shown by formation of bulbil-like propagules floating on water.

Thiogranum longum gen. nov., sp. nov., an obligately chemolithoautotrophic, sulfur-oxidizing bacterium of the family Ectothiorhodospiraceae isolated from a deep-sea hydrothermal field, and an emended description of the genus Thiohalomonas

A novel, obligately chemolithoautotrophic, sulfur-oxidizing bacterial strain, designated strain gps52(T), was isolated from a rock sample collected near the hydrothermal vents of the Suiyo Seamount in the Pacific Ocean. The cells possessed a Gram-stain-negative-type cell wall and contained menaquinone-8(H4) and menaquinone-9(H4) as respiratory quinones, and C16 : 1ω7c, C16 : 0 and C18 : 1ω7c as major cellular fatty acids. Neither storage compounds nor extensive internal membranes were observed in the cells. Strain gps52(T) grew using carbon dioxide fixation and oxidation of inorganic sulfur compounds with oxygen as electron acceptor. Optimal growth was observed at 32 °C, pH 6.5 and with 3 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain gps52(T) belongs to the family Ectothiorhodospiraceae and is different from any other known bacteria, with sequence similarities of less than 93 %. Based on phenotypic and phylogenetic findings, the isolate is considered to represent a novel genus and species in the family Ectothiorhodospiraceae, and the name Thiogranum longum gen. nov., sp. nov. is proposed. The type strain is gps52(T) ( = NBRC 101260(T) = DSM 19610(T)). An emended description of the genus Thiohalomonas is also proposed.

Diversity of aero-aquatic hyphomycetes from six streams in Doi Inthanon and Khao Yai tropical forests, Thailand

Abstract Randomly collected naturally decaying submerged/semi-submerged wood from six freshwater streams in two forests Doi Inthanon (DI) and Khao Yai National Parks (KY), Thailand was examined for aero-aquatic hyphomycetes following five field collections in July 2009 to November 2010. A total of 1,171 fungal occurrences belonging to 32 species in 13 genera were identified from DI (31%), and KY (28%), with 41 % common to both sites. Of these, eighteen were new records (56.25% of all species) for Thailand. Percentage occurrence of fungi at DI streams 1–3 were 30.77%, 35.99%, and 33.33%, respectively; while at streams 4–6 at KY, percentage occurrence was 31.11, 35.56, and 33.33%, respectively. The most common species at both forests (with >10% frequency of occurrence, FO) were Candelabrum brocchiatum (34.5%), followed by Pseudaegerita corticalis (15.4%), and Cancellidium applanatum (12.5%). These fungi were found at five streams in the two forests while Helicomyces roseus (6.3%), Candelabrum microsporum (4.3%) and Helicosporium panachaeum (4.3%) are regarded as common species. Seven species were regarded as rare, occurring only once per site with FO = 0.1% (Candelabrum sp., Helicoma atroseptatum, H. perelegans, H. resinae, Helicoma sp., Helicosporium aureum and H. gracile). A comparison of the fungal community at the two forests and six streams shows little difference in species composition.

Helicocentralis hyalina gen. et sp. nov., an aero-aquatic helicosporous fungus (Leotiomycetes, Ascomycota) in Thailand

During a survey of aero-aquatic fungi in Thailand, an undescribed helicosporous fungus was discovered. It is characterized by the presence of branched and septate hyphae with hyaline conidiophores that are micronematous or semi-macronematous, acrogenous and holoblastic. Conidia are hyaline and centrifugally coiled (excentric), helicoid or circinate, coiled 2–3 times clockwise or counterclockwise. After comparison of morphological and molecular characters with other aero-aquatic, helicosporous fungi, introduction of a new genus and species is proposed, Helicocentralis hyalina gen. et sp. nov. Phylogenetic analyses based on the combined sequence data from the small and large nuclear subunit ribosomal DNA (SSU and LSU), as well as internal transcribed spacer (ITS) rDNA sequence data, the fungus groups within the Leotiomycetes class with strong statistical support. The new genus is not related to other helicosporous hyphomycete genera (Helicoma, Helicomyces, Helicoön, and Helicosporium), which belong in the Tubeufiaceae, Dothideomycetes. Within the Leotiomycetes, our new fungus is distantly related to Helicodendron paradoxum (the type species). The new fungus is also compared morphologically to six similar helicosporous genera from Dothideomycetes and Leotiomycetes.

Sulfurovum denitrificans sp. nov., an obligately chemolithoautotrophic sulfur-oxidizing epsilonproteobacterium isolated from a hydrothermal field

A novel marine sulfur-oxidizing bacterium, designated strain eps51T, was isolated from a surface rock sample collected from the hydrothermal field of Suiyo Seamount on the Izu-Bonin Arc in the Western Pacific Ocean. This bacterium was Gram-staining-negative, non-motile and rod-shaped. Strain eps51T grew chemolithoautotrophically, by sulfur-oxidizing respiration with elemental sulfur and thiosulfate as electron donors and used only carbon dioxide as a carbon source. Oxygen and nitrate were used as its electron acceptors. The isolate grew optimally at 30 °C, at pH 7.0 and with 3 % NaCl. The predominant fatty acids were C16 : 1ω7c, C18 : 1ω7c and C16 : 0. The respiratory quinone was menaquinone-6 and the genomic DNA G+C content was 40.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequence revealed that eps51T represented a member of the genus Sulfurovum and the closest relative was Sulfurovum aggregans (96.7 %). Based on its phylogenetic position along with its physiological and chemotaxonomic characteristics, the name Sulfurovum denitrificans sp. nov. is proposed, with the type strain eps51T (=NBRC 102602T=DSM 19611T).