Akio Tonouchi

Methanobacterium kanagiense sp. nov., a hydrogenotrophic methanogen, isolated from rice-field soil

A pure culture of an obligately anaerobic, hydrogenotrophic, methanogenic archaeon, designated strain 169(T), which grows with hydrogen and carbon dioxide as the sole energy and carbon sources, was isolated from an anaerobic propionate-oxidizing enrichment culture originally obtained as an inoculant from rice-field soil in Japan. Cells of strain 169(T) were non-motile, Gram-reaction-variable and rod-shaped or slightly curved rods with rounded ends (1.6-5.0 × 0.35-0.5 µm). Strain 169(T) had fimbriae at both ends of the cell (up to ~10 per cell) but did not possess flagella. Ultrathin sections showed a single-layered, electron-dense cell wall about 6 nm thick, which is typical of Gram-positive bacteria. Growth was observed at 15 °C-45 °C (optimum 40 °C), at pH  6.5-9.6 (optimum pH 7.5-8.5) and in 0-70 g NaCl l(-1) (0-1.2 M) (optimum 5 g NaCl l(-1); 0.086 M). Strain 169(T) utilized only hydrogen and carbon dioxide as energy and carbon sources. The DNA G+C content was 39.3 mol%. The results of 16S rRNA gene sequence analysis indicated that strain 169(T) was most closely related to Methanobacterium subterraneum DSM 11074(T) (96.8 % sequence similarity) and Methanobacterium formicicum DSM 1535(T) (96.4 %). On the basis of its morphological, physiological and phylogenetic characteristics, strain 169(T) ( = DSM 22026(T) = JCM 15797(T)) represents a novel species of the genus Methanobacterium, for which the name Methanobacterium kanagiense sp. nov. is proposed.

Description of Anaerobacterium chartisolvens gen. nov., sp. nov., an obligately anaerobic bacterium from Clostridium rRNA cluster III isolated from soil of a Japanese rice field, and reclassification of Bacteroides cellulosolvens Murray et al. 1984 as Pseudobacteroides cellulosolvens gen. nov., comb. nov.

An obligately anaerobic bacterial strain designated T-1-35(T) was isolated as a dominant cultivable cellulose-degrading bacterium from soil of a Japanese rice field as an anaerobic filter-paper degrader. Cells of strain T-1-35(T) stained Gram-positive and were non-spore-forming rods with rounded ends, 0.8-1.0×3.5-15.0 µm, and motile by means of two to four polar flagella. Cells of strain T-1-35(T) exhibited pleomorphism: in aged cultures (over 90 days of incubation), almost all cells were irregularly shaped. Although no spore formation was observed, cells tolerated high temperatures, up to 90 °C for 10 min. The temperature range for growth was 15-40 °C, with an optimum at 35 °C. The pH range for growth was 5.5-9.0, with an optimum at pH 8.0-8.5 (slightly alkaliphilic). Strain T-1-35(T) fermented some carbohydrates to produce ethanol and lactate as the major products. Major cellular fatty acids were iso-C16 : 0 and iso-C13 : 0 3-OH. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain T-1-35(T) belonged to Clostridium rRNA cluster III. The closest relative of strain T-1-35(T) was Bacteroides cellulosolvens WM2(T), with 16S rRNA gene sequence similarity of 93.4 %. Phenotypic, physiological and molecular genetic methods demonstrated that strain T-1-35(T) was distinct from its phylogenetic relatives (members of Clostridium rRNA cluster III) because it predominantly produced ethanol, iso-C13 : 0 3-OH was a major cellular fatty acid and it always exhibited pleomorphism. On the basis of the results of a polyphasic taxonomic study, strain T-1-35(T) is considered to represent a novel genus and species, Anaerobacterium chartisolvens gen. nov., sp. nov. The type strain of Anaerobacterium chartisolvens is T-1-35(T) ( = DSM 27016(T) = NBRC 109520(T)). In addition, from the results of our phylogenetic analysis and its phenotypic features, the species Bacteroides cellulosolvens Murray et al. 1984 is proposed to be reclassified in the new genus Pseudobacteroides as Pseudobacteroides cellulosolvens gen. nov., comb. nov., with the type strain WM2(T) ( = ATCC 35603(T) = DSM 2933(T) = NRCC 2944(T)).

Aminivibrio pyruvatiphilus gen. nov., sp. nov., an anaerobic, amino-acid-degrading bacterium from soil of a Japanese rice field.

A novel anaerobic bacterium that could ferment amino acids and organic acids was isolated from an anaerobic, propionate-oxidizing enrichment culture originating from soil of a rice field in Japan. Cells of the isolate, designated strain 4F6E(T), were Gram-staining-negative, oxidase- and catalase-negative, non-spore-forming, vibrio-shaped, motile rods (0.8×2.0-2.5 µm) with two or three lateral flagella. Growth occurred at 20-42 °C (optimum at 37-40 °C), at pH 6.4-8.4 (optimum at pH 7.3) and at 0-1.5 % (w/v) NaCl (optimum at 0-0.5 %). Good growth occurred on glycine, serine, cysteine, pyruvate and citrate, whereas poor growth was observed on threonine, glutamine, L-malate, α-ketoglutarate, peptone and Casamino acids. In co-culture with the hydrogen-utilizing methanogen Methanobacterium formicicum JCM 10132(T), strain 4F6E(T) oxidized alanine, valine, leucine, isoleucine, methionine, aspartate, glutamate, histidine, asparagine and fumarate. Yeast extract was required for growth. The G+C content of genomic DNA was 61.9 mol%. A phylogenetic analysis based on comparison of the 16S rRNA gene sequence showed that the type strains of Fretibacterium fastidiosum, Aminobacterium colombiense and Aminobacterium mobile, members of the family Synergistaceae, were the closest relatives of strain 4F6E(T), with low sequence similarities (89.3, 89.5 and 86.2 %, respectively). Strain 4F6E(T) contained iso-C13 : 0 (24.43 %), iso-C15 : 0 (16.47 %) and C19 : 1ω11c/C19 : 1ω9c (16.32 %) as the major fatty acids, which differed from those of F. fastidiosum, Aminobacterium colombiense and Aminobacterium mobile. On the basis of phenotypic, chemotaxonomic and phylogenetic differences between strain 4F6E(T) and the type strains of F. fastidiosum and Aminobacterium species, we propose that strain 4F6E(T) represents a novel genus and species, Aminivibrio pyruvatiphilus gen. nov., sp. nov. The type strain of Aminivibrio pyruvatiphilus is strain 4F6E(T) ( = JCM 18417(T) = DSM 25964(T)).

Anaerobic 2-Propanol Degradation in Anoxic Paddy Soil and the Possible Role of Methanogens in Its Degradation

The anaerobic degradation of 2-propanol in anoxic paddy soil was studied with soil cultures and a 2-propanol-utilizing methanogen. Acetone was the first and the major intermediate involved in the methanogenic degradation of 2-propanol. Analyses with a methanogenesis inhibitor, bacteria antibiotics, and the addition of H2 to the gas phase revealed that 2-propanol oxidation to acetone directly occurred using 2-propanol-utilizing methanogens, but not with H2-producing syntrophic bacteria, for which the removal of acetone is required for complete 2-propanol oxidation. The 2-propanol-utilizing strain IIE1, which is phylogenetically closely related to Methanoculleus palmolei, was isolated from paddy soil, and the potential role of the strain in 2-propanol degradation was investigated. 2-Propanol is one of the representative fermentation intermediates in anaerobic environments. This is the first report on the anaerobic 2-propanol degradation process.

Cloning of the Gene Encoding an Endo-Acting Pectate Lyase from Streptomyces thermocarboxydus

An endo-acting family 3 pectate lyase (PL I) gene from Streptomyces thermocarboxydus was cloned and expressed in Escherichia coli. The deduced PL I sequence had highest similarity to a putative pectate lyase of S. coelicolor. Recombinant PL I had significantly lower specific activity, probably a result of incorrect folding, as indicated by circular dichroism spectra analysis.

Characterization of cultivable methanotrophs from paddy soils and rice roots

Abstract Characterization of methanotrophs isolated from paddy soils and rice (Oryza sativa) roots was investigated in the present study. The number of methanotrophs in root homogenates of the rice cultivar Mutsuhomare was 4.9 × 107 most-probable-number (MPN) g−1 dry roots, in Yumeakari it was 2.0 × 108 MPN g−1 dry roots and in Kirara it was 4.6 × 107 MPN g−1 dry roots. Although bacterial cells were observed infrequently on the surface and in the interiors of roots before incubation, a large number of colonies, measuring 0.5–5 mm in diameter, were observed on the sterilized roots after incubation on nitrate mineral agar plates under methane in air. In particular, a large number of colonies were observed at the emergence sites of lateral roots and root hairs. Strains MD5-1 and M1 were isolated from the roots of Mutsuhomare and strain R62 was isolated from the root homogenate of Yumeakari. All isolates were catalase-positive and oxidase-positive, Gram-negative, straight-rod-shaped and curved-rod-shaped bacteria, and formed exospores. The isolates were able to fix nitrogen and grew in the absence of copper. In addition, all were found to be positive for naphthalene-oxidizing activities (corresponding to soluble methane mono-oxygenase activities). Strains MD5-1, M1 and R62 were closely related to Methylosinus sporium. Methanotrophic strains W3-6, SD3-5 and 2-19, isolated previously from paddy field soils, were classified into Methylosinus (W3-6) and Methylocystis (SD3-5 and 2-19) type II methanotrophs. Isolates from the rice roots (MD5-1, R62 and M1) grew logarithmically when casamino acid was used as the nitrogen source; however, the growth of these strains was reduced on the nitrate medium. These strains preferred amino acids over inorganic nitrogen as a nitrogen source for growth.

Isolation and Characterization of a Novel Facultative Anaerobic Filamentous Fungus from Japanese Rice Field Soil

A novel filamentous fungus strain designated RB-1 was isolated into pure culture from Japanese rice field soil through an anaerobic role tube technique. The strain is a mitosporic fungus that grows in both aerobic and strict anaerobic conditions using various mono-, di-, tri-, and polysaccharides with acetate and ethanol productions. The amount of acetate produced was higher than that of ethanol in both aerobic and anaerobic cultures. The characteristic verrucose or punctuate conidia of RB-1 closely resembled those of some strains of the genus Thermomyces, a thermophilic or mesophilic anamorphic ascomycete. However, based on phylogenetic analysis with the small subunit (SSU) and large subunit (LSU) rDNA sequences, RB-1 was characterized as a member of the class Lecanoromycetes of the phylum Ascomycota. Currently, RB-1 is designated as an anamorphic ascomycete and is phylogenetically considered an incertae sedis within the class Lecanoromycetes.

Characteristics of an Anaerobic, Syntrophic, Butyrate-degrading Bacterium in Paddy Field Soil

The number of syntrophic butyrate-degrading bacte- ria in a flooded paddy field soil was 1.7×103 MPN/g dry soil. Butyrate was degraded to acetate and methane when paddy soils were incubated anaerobically with the addition of butyrate. However, butyrate degradation was completely suppressed by the addition of the specific inhibitor of methanogenesis, 2-bromoethanesulfonate (BES) to the soil. A hydrogen-using methanogen, strain TM-8, was isolated from flooded paddy field soil. Strain TM-8 was identified as Methanobacterium formicicum based on its physiology and phylogeny. Syntrophic butyrate-degrading bacteria were enumerated and isolated using strain TM-8. A syntrophic butyrate-degrading bacterium, strain TB-6, was isolated in coculture with strain TM-8 from paddy soil. The strain was Gram-negative, had curved rods, and grew on crotonate. Sulfate was not used as an electron acceptor. Strain TB-6 was closely related to S. wolfei subsp. wolfei. The relation between strain TB-6 and the members of Syntrophomonas are discussed.

Genetic polymorphisms in Japanese fragrant landraces and novel fragrant allele domesticated in northern Japan

Rice fragrance is an important characteristic for Southeast Asian consumers, and fragrant landraces from Japan were first recorded in the 17th century. Principal component analysis clearly showed that Japanese fragrant landraces were genetically different from non-Japanese fragrant landraces. Japanese fragrant landraces were composed of six clades, none of which carried the most common fragrance mutation, an 8-bp deletion in exon 7 of Badh2. Fragrant landraces comprised two major groups carrying different Badh2 mutations. One group carried a known SNP at exon13 and the other a SNP at the exon1-intron1 junction as splicing donor site. The latter was considered to be a potential splicing mutant group as a novel allele at Badh2. Heterozygosity (He) scores in the two fragrant groups were not significantly different from non-fragrant landraces and modern cultivars. However, lower He scores were found around the Badh2 locus in the two groups. The potential splicing mutant group showed a more extended haplotype than the E13 SNP group. A likely causal factor responsible for loss of function is a novel splicing mutation allele that may have been generated quite recently. The fragrance allele has dispersed as a result of out-crossing under local environmental conditions.

Clostridium oryzae sp. nov., from soil of a Japanese rice field.

An obligately anaerobic bacterial strain designated KC3(T) was isolated from a rice straw-degrading culture, for which soil of a Japanese rice field was used as the inoculum. Cells of strain KC3(T) were determined to be non-cellulolytic, Gram-stain-positive, non-motile, ellipsoidal, spore-forming rods, 0.8-1×4-25 µm. Endospores were formed at a terminal position in elongated cells (12-25 µm, mean 15 µm). The temperature range for growth was 20-50 °C, with an optimum at 37 °C. The pH range for growth was 5.0-7.5, with an optimum at pH 6.0 (slightly acidophilic). Strain KC3(T) fermented cellobiose to lactate, butyrate, acetate, formate, hydrogen and carbon dioxide. The major cellular fatty acids (>10 %) were C14 : 0, C16 : 0 and C19 : 0 cyclo 11,12 dimethylacetal. The DNA G+C content of strain KC3(T) was 37.5 mol%. 16S rRNA gene sequence analysis revealed that strain KC3(T) shared low sequence similarity (<93 %) with type strains of the genus Clostridium sensu stricto (Clostridium rRNA cluster I). Analyses of the DNA gyrase A and ATP synthase beta subunit sequences supported the affiliation of strain KC3(T) to the genus Clostridium sensu stricto. The evidence presented here indicates that strain KC3(T) represents a novel species of the genus Clostridium, for which the name Clostridium oryzae sp. nov. is proposed. The type strain of Clostridium oryzae is KC3(T) ( = DSM 28571(T) = NBRC 110163(T)).