Takao Iino

Candidatus Methanogranum caenicola: a Novel Methanogen from the Anaerobic Digested Sludge, and Proposal of Methanomassiliicoccaceae fam. nov. and Methanomassiliicoccales ord. nov., for a Methanogenic Lineage of the Class Thermoplasmata

The class Thermoplasmata harbors huge uncultured archaeal lineages at the order level, so-called Groups E2 and E3. A novel archaeon Kjm51a affiliated with Group E2 was enriched from anaerobic sludge in the present study. Clone library analysis of the archaeal 16S rRNA and mcrA genes confirmed a unique archaeal population in the enrichment culture. The 16S rRNA gene-based phylogeny revealed that the enriched archaeon Kjm51a formed a distinct cluster within Group E2 in the class Thermoplasmata together with Methanomassiliicoccus luminyensis B10T and environmental clone sequences derived from anaerobic digesters, bovine rumen, and landfill leachate. Archaeon Kjm51a showed 87.7% 16S rRNA gene sequence identity to the closest cultured species, M. luminyensis B10T, indicating that archaeon Kjm51a might be phylogenetically novel at least at the genus level. In fluorescence in situ hybridization analysis, archaeon Kjm51a was observed as coccoid cells completely corresponding to the archaeal cells detected, although bacterial rod cells still coexisted. The growth of archaeon Kjm51a was dependent on the presence of methanol and yeast extract, and hydrogen and methane were produced in the enrichment culture. The addition of 2-bromo ethanesulfonate to the enrichment culture completely inhibited methane production and increased hydrogen concentration, which suggested that archaeon Kjm51a is a methanol-reducing hydrogenotrophic methanogen. Taken together, we propose the provisional taxonomic assignment, named Candidatus Methanogranum caenicola, for the enriched archaeon Kjm51a belonging to Group E2. We also propose to place the methanogenic lineage of the class Thermoplasmata in a novel order, Methanomassiliicoccales ord. nov.

Complete Genome of Ignavibacterium album, a Metabolically Versatile, Flagellated, Facultative Anaerobe from the Phylum Chlorobi

Prior to the recent discovery of Ignavibacterium album (I. album), anaerobic photoautotrophic green sulfur bacteria (GSB) were the only members of the bacterial phylum Chlorobi that had been grown axenically. In contrast to GSB, sequence analysis of the 3.7-Mbp genome of I. album shows that this recently described member of the phylum Chlorobi is a chemoheterotroph with a versatile metabolism. I. album lacks genes for photosynthesis and sulfur oxidation but has a full set of genes for flagella and chemotaxis. The occurrence of genes for multiple electron transfer complexes suggests that I. album is capable of organoheterotrophy under both oxic and anoxic conditions. The occurrence of genes encoding enzymes for CO2 fixation as well as other enzymes of the reductive TCA cycle suggests that mixotrophy may be possible under certain growth conditions. However, known biosynthetic pathways for several amino acids are incomplete; this suggests that I. album is dependent upon on exogenous sources of these metabolites or employs novel biosynthetic pathways. Comparisons of I. album and other members of the phylum Chlorobi suggest that the physiology of the ancestors of this phylum might have been quite different from that of modern GSB.

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.

Methanospirillum lacunae sp. nov., a methane-producing archaeon isolated from a puddly soil, and emended descriptions of the genus Methanospirillum and Methanospirillum hungatei.

A mesophilic, hydrogenotrophic methanogen, designated strain Ki8-1(T), was isolated from soil. Cells were strictly anaerobic, Gram-stain-negative, non-sporulating, motile by means of a single flagellum or tufted flagella, and curved or wavy rod-shaped (11-25 μm long). The temperature and pH for optimum growth were 30 °C and 7.5. The strain grew best in basal medium without the addition of NaCl. Methane was produced from H(2) and formate. Acetate or yeast extract was required for growth. The G+C content of the genomic DNA of strain Ki8-1(T) was 45.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Ki8-1(T) was a member of the genus Methanospirillum and showed 95.1% sequence similarity to Methanospirillum hungatei NBRC 100397(T). On the basis of its phenotypic characteristics and phylogenetic position, strain Ki8-1(T) is considered to represent a novel species of the genus Methanospirillum, for which the name Methanospirillum lacunae sp. nov. is proposed. The type strain is Ki8-1(T) (NBRC 104920(T) =JCM 16384(T) =DSM 22751(T)). Emended descriptions of the genus Methanospirillum and of Methanospirillum hungatei are also provided.

Calditerrivibrio nitroreducens gen. nov., sp. nov., a thermophilic, nitrate-reducing bacterium isolated from a terrestrial hot spring in Japan.

A moderately thermophilic, nitrate-reducing bacterium, strain Yu37-1(T), was isolated from hot spring water from Yumata, Nagano, Japan. Cells of strain Yu37-1(T) were strictly anaerobic, Gram-negative, non-sporulating, motile by means of a single polar flagellum, vibrio-shaped and 1.4-2.0 microm long. The temperature and pH for optimum growth were 55 degrees C and pH 7.0-7.5, respectively. Strain Yu37-1(T) grew best in basal medium without the addition of NaCl. Acetate, pyruvate, lactate, fumarate, succinate, malate, yeast extract, peptone and Casamino acids were utilized as electron donors, with nitrate as the only electron acceptor. Ammonium was the end product from nitrate. The G+C content of the genomic DNA was 35.1 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that strain Yu37-1(T) could be accommodated in the family Deferribacteraceae and that its closest neighbours were members of the five genera of the family Deferribacteraceae, namely Deferribacter, Denitrovibrio, Flexistipes, Geovibrio and Mucispirillum, with similarities of only 83.2-86.2 %. The growth temperature and salinity range for growth of strain Yu37-1(T) differed from those of the phylogenetically related organisms. On the basis of phenotypic features and phylogenetic position, a novel genus and species are proposed, Calditerrivibrio nitroreducens gen. nov., sp. nov. Strain Yu37-1(T) (=NBRC 101217(T) =DSM 19672(T)) is the type strain of Calditerrivibrio nitroreducens.

Iron Corrosion Induced by Nonhydrogenotrophic Nitrate-Reducing Prolixibacter sp. Strain MIC1-1

ABSTRACT Microbiologically influenced corrosion (MIC) of metallic materials imposes a heavy economic burden. The mechanism of MIC of metallic iron (Fe0) under anaerobic conditions is usually explained as the consumption of cathodic hydrogen by hydrogenotrophic microorganisms that accelerates anodic Fe0 oxidation. In this study, we describe Fe0 corrosion induced by a nonhydrogenotrophic nitrate-reducing bacterium called MIC1-1, which was isolated from a crude-oil sample collected at an oil well in Akita, Japan. This strain requires specific electron donor-acceptor combinations and an organic carbon source to grow. For example, the strain grew anaerobically on nitrate as a sole electron acceptor with pyruvate as a carbon source and Fe0 as the sole electron donor. In addition, ferrous ion and l-cysteine served as electron donors, whereas molecular hydrogen did not. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MIC1-1 was a member of the genus Prolixibacter in the order Bacteroidales. Thus, Prolixibacter sp. strain MIC1-1 is the first Fe0-corroding representative belonging to the phylum Bacteroidetes. Under anaerobic conditions, Prolixibacter sp. MIC1-1 corroded Fe0 concomitantly with nitrate reduction, and the amount of iron dissolved by the strain was six times higher than that in an aseptic control. Scanning electron microscopy analyses revealed that microscopic crystals of FePO4 developed on the surface of the Fe0 foils, and a layer of FeCO3 covered the FePO4 crystals. We propose that cells of Prolixibacter sp. MIC1-1 accept electrons directly from Fe0 to reduce nitrate.

First cultivation and ecological investigation of a bacterium affiliated with the candidate phylum OP5 from hot springs.

ABSTRACT The phylogenetic group termed OP5 was originally discovered in the Yellowstone National Park hot spring and proposed as an uncultured phylum; the group was afterwards analyzed by applying culture-independent approaches. Recently, a novel thermophilic chemoheterotrophic filamentous bacterium was obtained from a hot spring in Japan that was enriched through various isolation procedures. Phylogenetic analyses of the isolate have revealed that it is closely related to the OP5 phylum that has mainly been constructed with the environmental clones retrieved from thermophilic and mesophilic anaerobic environments. It appears that the lineage is independent at the phylum level in the domain Bacteria. Therefore, we designed a primer set for the 16S rRNA gene to specifically target the OP5 phylum and performed quantitative field analysis by using the real-time PCR method. Thus, the 16S rRNA gene of the OP5 phylum was detected in some hot-spring samples with the relative abundance ranging from 0.2% to 1.4% of the prokaryotic organisms detected. The physiology of the above-mentioned isolate and the related environmental clones indicated that they are scavengers contributing to the sulfur cycle in nature.

Dysgonomonas termitidis sp. nov., isolated from the gut of the subterranean termite Reticulitermes speratus.

A facultatively anaerobic, Gram-stain-negative, non-motile and rod-shaped bacterium, strain N-10(T), was isolated from the gut of the termite Reticulitermes speratus. Strain N-10(T) was closely related to Dysgonomonas gadei JCM 16698(T) according to 16S rRNA gene sequence similarity analysis (98 %) and DNA-DNA relatedness value (≤61.3 %). The optimum growth temperature of strain N-10(T) was 30 °C, which was distinct from that (37 °C) of known species of the genus Dysgonomonas. Growth of strain N-10(T) was inhibited on medium containing 5 or 20 % bile, unlike other species of the genus Dysgonomonas. In addition, acid production in the API 20A system and enzymic reactions in the Rapid ID 32A system of strain N-10(T) differed from those of other species of the genus Dysgonomonas. Based on these characteristics, strain N-10(T) represents a novel species of the genus Dysgonomonas, for which the name Dysgonomonas termitidis sp. nov. is proposed. The type strain is N-10(T) ( = JCM 30204(T) = CCUG 66188(T)).

Description of Mariniphaga anaerophila gen. nov., sp. nov., a facultatively aerobic marine bacterium isolated from tidal flat sediment, reclassification of the Draconibacteriaceae as a later heterotypic synonym of the Prolixibacteraceae and description of the family Marinifilaceae fam. nov.

A mesophilic, chemoheterotrophic bacterium, strain Fu11-5(T), was isolated from tidal-flat sediment from Tokyo Bay, Chiba, Japan. Cells of strain Fu11-5(T) were facultatively aerobic, Gram-negative, non-sporulating, non-motile and rod-shaped (1.9-6.9 µm long). Strain Fu11-5(T) grew optimally at 35-37 °C and pH 6.5-7.0 and with 1-2% (w/v) NaCl. Oxygen and l-cysteine were used as an alternative electron acceptor and donor, respectively. Strain Fu11-5(T) also grew fermentatively on some pentoses, hexoses and disaccharides and soluble starch. Succinic acid was the major end product from d-glucose. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain Fu11-5(T) was affiliated with the order Bacteroidales, and its nearest neighbours were members of the genera Meniscus, Prolixibacter, Sunxiuqinia, Mangrovibacterium and Draconibacterium, with 87-91% sequence similarity. Cell morphology, optimum growth temperature and utilization of sugars of strain Fu11-5(T) distinguished the strain from phylogenetically related bacteria. On the basis of its phenotypic features and phylogenetic position, a novel genus and species are proposed to accommodate strain Fu11-5(T), with the name Mariniphaga anaerophila gen. nov., sp. nov. The type strain of Mariniphaga anaerophila is strain Fu11-5(T) ( =JCM 18693(T) =NBRC 109408(T) =DSM 26910(T)). We also propose to combine the family Draconibacteriaceae into the family Prolixibacteraceae as a later heterotypic synonym and to place the distinct sublineage of the genus Marinifilum in the family Marinifilaceae fam. nov.

Gluconacetobacter kakiaceti sp. nov., an acetic acid bacterium isolated from a traditional Japanese fruit vinegar

Two novel acetic acid bacteria, strains G5-1(T) and I5-1, were isolated from traditional kaki vinegar (produced from fruits of kaki, Diospyros kaki Thunb.), collected in Kumamoto Prefecture, Japan. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains G5-1(T) and I5-1 formed a distinct subline in the genus Gluconacetobacter and were closely related to Gluconacetobacter swingsii DST GL01(T) (99.3% 16S rRNA gene sequence similarity). The isolates showed 96-100% DNA-DNA relatedness with each other, but <53% DNA-DNA relatedness with closely related members of the genus Gluconacetobacter. The isolates could be distinguished from closely related members of the genus Gluconacetobacter by not producing 2- and 5-ketogluconic acids from glucose, producing cellulose, growing without acetic acid and with 30% (w/v) d-glucose, and producing acid from sugars and alcohols. Furthermore, the genomic DNA G+C contents of strains G5-1(T) and I5-1 were a little higher than those of their closest phylogenetic neighbours. On the basis of the phenotypic characteristics and phylogenetic position, strains G5-1(T) and I5-1 are assigned to a novel species, for which the name Gluconacetobacter kakiaceti sp. nov. is proposed; the type strain is G5-1(T) (=JCM 25156(T)=NRIC 0798(T)=LMG 26206(T)).