Search

H2 is an important fermentation intermediate in anaerobic environments. Although H2 occurs at very low partial pressures in the environments, the culture and isolation of H2-utilizing microorganisms is usually carried out under very high H2 pressures, which might have hampered the discovery and understanding of microorganisms adapting to low H2 environments. Here we constructed a culture system designated the “iron corrosion-assisted H2-supplying (iCH) system” by connecting the gas phases of two vials (one for the iron corrosion reaction and the other for culturing microorganisms) to achieve cultures of microorganisms under low H2 pressures. We conducted enrichment cultures for methanogens and acetogens using rice paddy field soil as the microbial source. In the enrichment culture of methanogens under canonical high H2 pressures, only Methanobacterium spp. were enriched. By contrast, Methanocella spp. and Methanoculleus spp., methanogens adapting to low H2 pressures, were specifically enriched in the iCH cultures. We also observed selective enrichment of acetogen species by the iCH system (Acetobacterium spp. and Sporomusa spp.), whereas Clostridium spp. predominated in the high H2 cultures. These results demonstrate that the iCH system facilitates culture of anaerobic microorganisms under low H2 pressures, which will enable the selective culture of microorganisms adapting to low H2 environments.

The origin of eukaryotes remains unclear1–4. Current data suggest that eukaryotes may have emerged from an archaeal lineage known as ‘Asgard’ archaea5,6. Despite the eukaryote-like genomic features that are found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear, owing to the lack of cultured representatives and corresponding physiological insights. Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon—‘Candidatus Prometheoarchaeum syntrophicum’ strain MK-D1—is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexes have been proposed for Asgard archaea6, the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle–engulf–endogenize (also known as E3) model., Isolation and characterization of an archaeon that is most closely related to eukaryotes reveals insights into how eukaryotes may have evolved from prokaryotes.

A strictly aerobic, bacteriochlorophyll a-containing betaproteobacterium, designated strain W35T, was isolated from a biofilm sampled at Tama River in Japan. The non-motile and rod-shaped cells formed pink-beige pigmented colonies on agar plates containing organic compounds, and showed an in vivo absorption maximum at 871 nm in the near-infrared region, typical for the presence of bacteriochlorophyll a. The new bacterial strain is Gram-negative, and oxidase- and catalase-positive. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain W35T was closely related to species in the genus Aquabacterium. The closest phylogenetic relatives of strain W35T were Aquabacterium commune B8T (97.9 % sequence similarity), Aquabacterium citratiphilum B4T (97.2 %) and Aquabacterium limnoticum ABP-4T (97.0 %). The major cellular fatty acids were C16 : 1ω7c (50.4 %), C16 : 0 (22.7 %), summed feature 8 (C18 : 1ω7c/C18 : 1ω6c; 9.7 %), C18 : 3ω6c (5.5 %), C12 : 0 (5.3 %) and C10 : 0 3OH (2.7 %). The respiratory quinone was ubiquinone-8. Predominant polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The G+C content of the genomic DNA was 70.4 mol% (genome data) and 71.4 mol% (HPLC). The genome size of strain W35T is 6.1 Mbp and average nucleotide identity analysis indicated genome similarities of strain W35T and related Aquabacterium type strains to be 78-79 %. The results of polyphasic comparisons showed that strain W35T was clearly distinguishable from other members of the genus Aquabacterium. Therefore, we propose a new species in the genus Aquabacterium, namely, Aquabacterium pictum sp. nov. The type strain is W35T (=DSM 106757T=NBRC 111963T). The description of the genus Aquabacterium is also emended.

Methane-generating archaea drive the final step in anaerobic organic compound mineralization and dictate the carbon flow of Earth’s diverse anoxic ecosystems in the absence of inorganic electron acceptors. Although such Archaea were presumed to be restricted to life on simple compounds like hydrogen (H2), acetate or methanol, an archaeon, Methermicoccus shengliensis, was recently found to convert methoxylated aromatic compounds to methane. Methoxylated aromatic compounds are important components of lignin and coal, and are present in most subsurface sediments. Despite the novelty of such a methoxydotrophic archaeon its metabolism has not yet been explored. In this study, transcriptomics and proteomics reveal that under methoxydotrophic growth M. shengliensis expresses an O-demethylation/methyltransferase system related to the one used by acetogenic bacteria. Enzymatic assays provide evidence for a two step-mechanisms in which the methyl-group from the methoxy compound is (1) transferred on cobalamin and (2) further transferred on the C1-carrier tetrahydromethanopterin, a mechanism distinct from conventional methanogenic methyl-transfer systems which use coenzyme M as final acceptor. We further hypothesize that this likely leads to an atypical use of the methanogenesis pathway that derives cellular energy from methyl transfer (Mtr) rather than electron transfer (F420H2 re-oxidation) as found for methylotrophic methanogenesis.

We previously showed that novel filterable bacteria remain in “sterile” (&lt, 0.2 μm filtered) terrestrial environmental samples from Japan, China, and Arctic Norway. Here, we characterized the novel filterable strain IZ6T, a representative strain of a widely distributed lineage. Phylogenetic analysis showed that this strain was affiliated with the Rhizobiales (now proposed as Hyphomicrobiales) of Alphaproteobacteria, but distinct from any other type strains. Strain IZ6T shared the following chemotaxonomic features with the closest (but distantly) related type strain, Flaviflagellibacter deserti SYSU D60017T: ubiquinone-10 as the major quinone, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol as major polar lipids, and slightly high G+C content of 62.2 mol%. However, the cellular fatty acid composition differed between them, and the unsaturated fatty acid (C18:1ω7c/C18:1ω6c) was predominantly found in our strain. Moreover, unlike methyrotrophs and nitrogen-fixers of the neighboring genera of Hyphomicrobiales (Rhizobiales), strain IZ6T cannot utilize a one-carbon compound (e.g., methanol) and fix atmospheric nitrogen gas. These findings were consistent with the genome-inferred physiological potential. Based on the phylogenetic, physiological, and chemotaxonomic traits, we propose that strain IZ6T represents a novel genus and species with the name Terrihabitans soli gen. nov., sp. nov. (=NBRC 106741T = NCIMB 15058T). The findings will provide deeper insight into the eco-physiology of filterable microorganisms.

We report the complete genome sequence of Lactobacillus helveticus JCM 1004, an aminopeptidase-producing lactic acid bacterium. The genome consists of a circular chromosome which comprises 2,261,280 bp, with a G+C content of 37.56%. The genome was predicted to harbor 13 rRNA genes, 64 tRNA genes, and 2,462 protein-coding sequences.

Atopobiaceae sp. strain P1 ( Actinobacteria , Coriobacteriales ) was isolated from mouse feces. Here, we report the complete genome sequence of this strain, which has a total size of 2,028,478 bp and a G+C content of 58.6%.

A novel slow-growing, facultatively anaerobic, filamentous bacterium, strain MO-CFX2T, was isolated from a methanogenic microbial community in a continuous-flow bioreactor that was established from subseafloor sediment collected off the Shimokita Peninsula of Japan. Cells were multicellular filamentous, non-motile and Gram-stain-negative. The filaments were generally more than 20 µm (up to approximately 200 µm) long and 0.5–0.6 µm wide. Cells possessed pili-like structures on the cell surface and a multilayer structure in the cytoplasm. Growth of the strain was observed at 20–37 °C (optimum, 30 °C), pH 5.5–8.0 (pH 6.5–7.0), and 0–30 g l−1 NaCl (5 g l−1 NaCl). Under optimum growth conditions, doubling time and maximum cell density were estimated to be approximately 19 days and ~105 cells ml−1, respectively. Strain MO-CFX2T grew chemoorganotrophically on a limited range of organic substrates in anaerobic conditions. The major cellular fatty acids were saturated C16 : 0 (47.9 %) and C18 : 0 (36.9 %), and unsaturated C18 : 1ω9c (6.0 %) and C16 : 1ω7 (5.1 %). The G+C content of genomic DNA was 63.2 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain MO-CFX2T shares a notably low sequence identity with its closest relatives, which were Thermanaerothrix daxensis GNS-1T and Thermomarinilinea lacunifontana SW7T (both 85.8 % sequence identity). Based on these phenotypic and genomic properties, we propose the name Aggregatilinea lenta gen. nov., sp. nov. for strain MO-CFX2T (=KCTC 15625T, =JCM 32065T). In addition, we also propose the associated family and order as Aggregatilineaceae fam. nov. and Aggregatilineales ord. nov., respectively.

The present study characterized the interactions of microbial populations in activated sludge systems during the operational period after an increase in the wastewater flow rate and consequential ammonia accumulation using a 16S rRNA gene sequencing-based network analysis. Two hundred microbial populations accounting for 81.8% of the total microbiome were identified. Based on a co-occurrence analysis, Nitrosomonas-type ammonia oxidizers had one of the largest number of interactions with diverse bacteria, including a bulking-associated Thiothrix organism. These results suggest that an increased flow rate has an impact on constituents by changing ammonia concentrations and also that Nitrosomonas- and Thiothrix-centric responses are critical for ammonia removal and microbial community recovery.

Primordial microorganisms are postulated to have emerged in H2-rich alkaline Hadean serpentinite-hosted environments with homoacetogenesis as a core metabolism. However, investigation of two modern serpentinization-active analogues of early Earth reveals that conventional H2-/CO2-dependent homoacetogenesis is thermodynamically unfavorable in situ due to picomolar CO2 levels. Through metagenomics and thermodynamics, we discover unique taxa capable of metabolism adapted to the habitat. This included a novel deep-branching phylum, “Ca. Lithoacetigenota”, that exclusively inhabits Hadean analogues and harbors genes encoding alternative modes of H2-utilizing lithotrophy. Rather than CO2, these metabolisms utilize reduced carbon compounds detected in situ presumably serpentinization-derived: formate and glycine. The former employs a partial homoacetogenesis pathway and the latter a distinct pathway mediated by a rare selenoprotein – the glycine reductase. A survey of serpentinite-hosted system microbiomes shows that glycine reductases are diverse and nearly ubiquitous in Hadean analogues. “Ca. Lithoacetigenota” glycine reductases represent a basal lineage, suggesting that catabolic glycine reduction is an ancient bacterial innovation for gaining energy from geogenic H2 even under serpentinization-associated hyperalkaline, CO2-poor conditions. This draws remarkable parallels with ancestral archaeal H2-driven methyl-reducing methanogenesis recently proposed. Unique non-CO2-reducing metabolic strategies presented here may provide a new view into metabolisms that supported primordial life and the diversification of LUCA towards Archaea and Bacteria.

Anaerostipes caccae strain L1-92T is a well-known butyrate-producing bacterium that has been isolated from human feces. In this announcement, we present the complete genome sequence of A. caccae strain L1-92T, which comprises 3,590,719 bp with a G+C content of 44.30%. The genome harbors 3,369 predicted protein-coding genes.

A novel mesophilic sulfate-reducing bacterium, strain HN2T, was isolated from groundwater sampled from the subsurface siliceous mudstone of the Wakkanai Formation located in Horonobe, Hokkaido, Japan. The bacterium was Gram-negative and vibrio-shaped, and its motility was conferred by a single polar flagellum. Cells had desulfoviridin. Catalase and oxidase activities were not detected. It grew in the temperature range of 25–40 °C (optimum, 35 °C) and pH range of 6.3–8.1 (optimum, pH 7.2–7.6). It used sulfate, thiosulfate, dimethyl sulfoxide, anthraquinone-2,6-disulfonate, Fe3+, and manganese oxide, but not elemental sulfur, nitrite, nitrate, or fumarate as electron acceptors. The strain showed weak growth with sulfite as the electron acceptor. Fermentative growth with pyruvate, lactate and cysteine was observed in the absence of sulfate, but not with malate or fumarate. NaCl was not required, but the strain tolerated up to 40 g l–1. Strain HN2T did not require vitamins. The major cellular fatty acids were iso-C15 : 0 (23.8 %), C18 : 1 ω9t (18.4 %), C18 : 0 (15.0 %), C16 : 0 (14.5 %), and anteiso-C17 :0 (10.1 %). The major respiratory quinone was menaquinone MK-6(H2). The G+C content of the genomic DNA was 56.7 mol%. Based on 16S rRNA gene sequence analysis, the closest phylogenetic relative of strain HN2T is Desulfovibrio psychrotolerans JS1T (97.0 %). Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values of the strains HN2T and D. psychrotolerans JS1T were 22.2 and 79.8 %, respectively. Based on the phenotypic and molecular genetic evidence, we propose a novel species, D. subterraneus sp. nov. with the type strain HN2T (=DSM 101010T=NBRC 112213T).

An anaerobic thermophilic, rod-shaped bacterium possessing a unique non-lipid sheathed-like structure enveloping a single-membraned cell, designated strain NRmbB1T was isolated from at the deep subsurface oil field located in Yamagata Prefecture, Japan. Growth occurred with 40-60°C (optimum, 55°C), 0-2% (2%), NaCl and pH 6.0-8.5 (8.0). Fermentative growth with various sugars was observed. Glucose-grown cells generated acetate, hydrogen, pyruvate and lactate as the main end products. Syntrophic growth occurred with glucose, pyruvate and 3,4,5-trimethoxybenzoate in the presence of an H2-scavenging partner, and growth on 3,4,5-trimethoxybenzoate was only observed under syntrophic condition. The predominant cellular fatty acids were C16:0, iso-C16:0, anteiso-C15:0, and iso-C14:0. Respiratory quinone was not detected. The genomic G+C content was 40.8mol%. Based on 16S rRNA gene phylogeny, strain NRmbB1T belongs to a distinct order-level clade in the class Clostridia of the phylum Firmicutes, sharing low similarity with other isolated organisms (i.e., 87.5% for top hit Moorella thermoacetica DSM 2955T). In total, chemotaxonomic, phylogenetic and genomic characterization revealed that strain NRmbB1T (=KCTC 25035T, =JCM 39120T) represents a novel species of a new genus. In addition, we also propose the associated family and order as Koleobacteraceae fam. nov and Koleobacterales ord. nov., respectively.

Understanding the status of gut microbiota has been recognized as crucial in health management and disease treatment. To meet the demands of medical and biological applications where rapid evaluation of gut microbiota in limited research environment is essential, we developed new sensing systems able to readout the overall characteristics of complex microbiota. Response patterns generated by a synthetic library of 12 charged block-copolymers with aggregation-induced emission units were analyzed with pattern recognition algorithms, allowing to identify the species/phyla of 16 axenic cultures of intestinal bacterial strains. More importantly, our method clearly classified artificial models of obesity-associated gut microbiota, and further succeeded in detecting sleep disorders in mice through comparative analysis of the normal/abnormal mouse gut microbiota. Our techniques can analyze complex bacterial samples far more quickly, simply and inexpensively than common metagenome-based methods, offering a powerful and complementary tool for gut microbiome analysis for practical use, e.g., in clinical settings.

The microbial communities inhabiting the fronds of duckweeds have not been investigated in as much detail as those on the roots. We herein examined the microbial communities in three duckweed species using 16S rRNA amplicon sequencing and compared them to those on the roots. The microbial compositions of the fronds were distinct from those of the roots in the three species. Various types of taxonomic bacteria, including rarely cultivated phyla, Acidobacteria, Armatimonadetes, and Verrucomicrobia, were also isolated from the fronds, but at a slightly lower abundance than those from the roots. These results suggest that duckweed fronds are an alternative source for isolating rare and novel microbes, which may otherwise be recalcitrant to cultivation using conventional strategies.

We herein described a new microbial isolation method using the interaction between the floating aquatic plant, duckweed, and microbes. We harvested microbial cells from Japanese loosestrife roots and co-cultivated these cells with aseptic duckweed using artificial inorganic medium for the plant for four weeks. During the co-cultivation, some duckweeds were collected every week, and the roots were used for microbial isolation using a low-nutrient plate medium. As a result, diverse microbial isolates, the compositions of which differed from those of the original source (Japanese loosestrife root), were obtained when the roots of duckweed were collected after 2 weeks of cultivation. We also successfully isolated a wide variety of novel microbes, including two strains within the rarely cultivated phylum, Armatimonadetes. The present study shows that a duckweed-microbe co-cultivation approach together with a conventional technique (direct isolation from a microbial source) effectively obtains more diverse microbes from a sole environmental sample.

We report the draft genome sequence of a novel Rhodospirillales bacterium strain, TMPK1, isolated from a micropore-filtered soil suspension. This strain has a genome of 4,249,070 bp, comprising 4,151 protein-coding sequences. The genome sequence data further suggest that strain TMPK1 is an alphaproteobacterium capable of carotenoid production.

A number of novel bacteria including members of rarely cultivated phyla, Acidobacteria and Verrucomicrobia, were successfully isolated from the roots of two emergent plants, Iris pseudacorus and Scirpus juncoides, by a simple culturing method. A total of 47.1% (66 strains) for I. pseudacorus and 42.1% (59 strains) for S. juncoides of all isolates (140 strains from each sample) were phylogenetically novel. Furthermore, Acidobacteria and Verrucomicrobia occupied 10.7% (15 strains) and 2.9% (4 strains) of I. pseudacorus isolates, and 2.1% (3 strains) and 3.6% (5 strains) of S. juncoides isolates, respectively, indicating that plant roots are attractive sources for isolating rarely cultivated microbes.

A key feature that differentiates prokaryotes from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we report isolation of an anaerobic bacterium that possesses an additional intracytoplasmic membrane surrounding a nucleoid, affiliates with the yet-to-be-cultivated ubiquitous phylum ‘Ca. Atribacteria’, and possesses unique genomic features likely associated with organization of complex cellular structure. Exploration of the uncharted microorganism overturned the prevailing dogma of prokaryotic cell structure.

N-Acyl homoserine lactones (AHLs) are signaling molecules used in the quorum sensing (QS) of Gram-negative bacteria. Some bacteria interfere with the QS system using AHL-inactivating enzymes, commonly known as quorum-quenching (QQ) enzymes. We have recently isolated a new QQ bacterium showing high resistance to multiple β-lactam antibiotics, and its QQ enzyme (MacQ) confers β-lactam antibiotic resistance and exhibits QQ activities. This observation suggests the possibility of isolating novel QQ bacteria from β-lactam antibiotic-resistant bacteria. In this direction, we attempted to isolate penicillin G (PENG)-resistant bacteria from penicillin-contaminated river sediments and activated sludge treating penicillin-containing wastewater and characterize their QQ activities. Of 19 PENG-resistant isolates, six isolates showed high QQ activity toward a broad range of AHLs, including AHLs with 3-oxo substituents. Five of the six AHL-degraders showed AHL-acylase activity and hydrolyzed the amide bond of AHLs, whereas the remaining one strain did not show AHL-acylase activity, suggesting that this isolate may likely possess alternative degradation mechanism such as AHL-lactonase activity hydrolyzing the lactone ring of AHLs. The 16S rRNA gene sequence analysis results categorized these six AHL-degrading isolates into at least five genera, namely, Sphingomonas (Alphaproteobacteria), Diaphorobacter (Betaproteobacteria), Acidovorax (Betaproteobacteria), Stenotrophomonas (Gammaproteobacteria), and Mycobacterium (Actinobacteria); of these, Mycobacterium sp. M1 has never been known as QQ bacteria. Moreover, multiple β-lactam antibiotics showed high minimum inhibitory concentrations (MICs) when tested against all of isolates. These results strongly demonstrate that a wide variety of β-lactam antibiotic-resistant bacteria possess QQ activities. Although the genetic and enzymatic elements are yet unclear, this study may infer the functional and evolutionary correlation between β-lactam antibiotic resistance and QQ activities.

Duckweeds are small, fast growing, and starch- and protein-rich aquatic plants expected to be a next generation energy crop and an excellent biomaterial for phytoremediation. Despite such an importance, very little is known about duckweed–microbe interactions that would be a key biological factor for efficient industrial utilization of duckweeds. Here we first report the duckweed growth promoting ability of bacterial strains belonging to the phylum Acidobacteria, the members of which are known to inhabit soils and terrestrial plants, but their ecological roles and plant–microbe interactions remain largely unclear. Two novel Acidobacteria strains, F-183 and TBR-22, were successfully isolated from wild duckweeds and phylogenetically affiliated with subdivision 3 and 6 of the phylum, respectively, based on 16S rRNA gene sequence analysis. In the co-culture experiments with aseptic host plants, the F-183 and TBR-22 strains visibly enhanced growth (frond number) of six duckweed species (subfamily Lemnoideae) up to 1.8–5.1 times and 1.6–3.9 times, respectively, compared with uninoculated controls. Intriguingly, both strains also increased the chlorophyll content of the duckweed (Lemna aequinoctialis) up to 2.4–2.5 times. Under SEM observation, the F-183 and TBR-22 strains were epiphytic and attached to the surface of duckweed. Taken together, our findings suggest that indigenous plant associated Acidobacteria contribute to a healthy growth of their host aquatic plants.

Lactobacillus gasseri is one of the most likely probiotic candidates among many Lactobacillus species. Although bile salt resistance has been defined as an important criterion for selection of probiotic candidates since it allows probiotic bacteria to survive in the gut, both its capability and its related enzyme, bile salt hydrolase (BSH), in L. gasseri is still largely unknown. Here, we report that the well-known probiotic bacterium L. gasseri JCM1131T possesses BSH activity and bile salt resistance capability. Indeed, this strain apparently showed BSH activity on the plate assay and highly tolerated the primary bile salts and even taurine-conjugated secondary bile salt. We further isolated a putative BSH enzyme (LagBSH) from strain JCM1131T and characterized the enzymatic function. The purified LagBSH protein exhibited quite high deconjugation activity for taurocholic acid and taurochenodeoxycholic acid. The lagBSH gene was constitutively expressed in strain JCM1131T, suggesting that LagBSH likely contributes to bile salt resistance of the strain and may be associated with survival capability of strain JCM1131T within the human intestine by bile detoxification. Thus, this study first demonstrated the bile salt resistance and its responsible enzyme (BSH) activity in strain JCM1131T, which further supports the importance of the typical lactic acid bacterium as probiotics.

A mesophilic, hydrogenotrophic methanogen, designated strain MobHT, was isolated from sediments derived from deep sedimentary, natural-gas-bearing aquifers in Japan. Strain MobHT utilized H2/CO2 or formate, but not ethanol, 1-propanol, 2-propanol, 2-butanol or cyclopentanol, for growth and methane production. In addition, acetate and tungsten were required for growth. Yeast extract stimulated the growth, but was not required. The cells were weakly motile with multiple flagella, presented as a curved-rod-shaped (0.8×2.0 µm) and occurred singly or in pairs. Strain MobHT grew at 15–40 °C (optimum 35 °C) and at pH 5.9–7.9 (optimum pH 7.0–7.5). The sodium chloride range for growth was 0–5.8 % (optimum 2 %). The G+C content of the genomic DNA was 37.6 mol%. In the phylogenetic tree based on the 16S rRNA gene sequences, strain MobHT clustered together with Methanomicrobium mobile (95.4 % in sequence similarity), and formed a distinct clade from Methanolacinia petrolearia SEBR 4847T (95.6 %) and Methanolacinia paynteri G-2000T (95.4 %). The two species of the genus Methanolacinia utilized 2-propanol, whereas strain MobHT and Methanomicrobium mobile , the sole species of the genus Methanomicrobium , do not. Based on phenotypic and phylogenetic features, we propose a novel species for the isolate with the name, Methanomicrobium antiquum sp. nov. The type strain is MobHT (=DSM 21220T=NBRC 104160T).

Microbes make methane from coal Methane associated with coal beds is an important global resource of natural gas. Much of the methane in coal comes from microbial methanogenesis. Mayumi et al. characterized a strain of Methermicoccus shengliensis that, unexpectedly, is capable of making methane from the dozens of methoxylated aromatic compounds found in a variety of coal types (see the Perspective by Welte). Isotope tracer experiments showed that this organism could also incorporate carbon dioxide into methane. Science , this issue p. 222 ; see also p. 184

A novel, strictly anaerobic, short rod-shaped bacterium, designated strain TBC1T, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor treating high-strength starch-based organic wastewater. Cells of this strain were 2–4 µm long and 0.4–0.6 µm wide. They were non-motile and Gram-stain-negative. The optimum growth temperature was 30–37 °C, with a range of 20–40 °C. The optimum pH for growth was around pH 7.0, while growth occurred in a range of pH 6.5–9.0. Strain TBC1T grew chemo-organotrophically on a narrow range of carbohydrates under anaerobic conditions. Yeast extract was required for its growth. The major fermentative end products from glucose, supplemented with yeast extract, were acetate, malate, propionate, formate and hydrogen. Doubling time under optimal growth conditions was estimated to be 1 day. The DNA G+C content of strain TBC1T was 49.2 mol% as determined by HPLC. Major cellular fatty acids were C16 : 0, C18 : 0, C16 : 1 ω9c and C18 : 1 ω9c. Based on its 16S rRNA gene sequence, strain TBC1T was shown to represent a distinct lineage at the family level in the phylum Bacteroidetes . Among previously described species of this phylum, Mucilaginibacter boryungensis BDR-9T ( Sphingobacteriaceae ) displayed the highest sequence similarity (85.9 %) with strain TBC1T. Phylogenomic analyses using 38–83 single copy marker genes also supported the novelty of strain TBC1T at the family level. Based on its characteristics, strain TBC1T (=JCM 30898T=DSM 100618T) is considered to be the type strain of a novel species of a new genus, Lentimicrobium saccharophilum gen. nov., sp. nov. A new family, Lentimicrobiaceae fam. nov., is also proposed encompassing the strain and related environmental 16S rRNA gene clone sequences.

A novel obligately anaerobic bacterium, designated strain TC1T, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor treating high-strength starch-based wastewater. Cells had a multicellular filamentous morphology, stained Gram-negative and were non-motile. The filaments were flexible, generally >100 μm long and 0.3–0.4 μm wide. Growth of the isolate was observed at 25–43 °C (optimum 37 °C) and pH 6.0–8.5 (optimum pH 7.0). Strain TC1T grew chemo-organotrophically on a range of carbohydrates under anaerobic conditions. Yeast extract was required for growth. The major fermentative end products of glucose, supplemented with yeast extract, were acetate, lactate, succinate, propionate, formate and hydrogen. Co-cultivation with the hydrogenotrophic methanogen Methanospirillum hungatei DSM 864T enhanced growth of the isolate. The DNA G+C content was determined experimentally to be 42.1 mol%. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0 and iso-C17 : 0 3-OH. Based on 16S rRNA gene sequence analysis, strain TC1T belonged to the class Anaerolineae in the phylum Chloroflexi, in which Ornatilinea apprima P3M-1T was its closest phylogenetic relative (88.3 % nucleotide identity). Phylogenomic analyses using 38 and 83 single-copy marker genes also supported the novelty of strain TC1T at least at the genus level. Based on phylogenetic, genomic and phenotypic characteristics, we propose that strain TC1T represents a novel species of a new genus, for which we suggest the name Flexilinea flocculi gen. nov., sp. nov. The type strain of Flexilinea flocculi is strain TC1T ( = JCM 30897T = CGMCC 1.5202T).

High-affinity H2 -oxidizing bacteria possessing group 5 [NiFe]-hydrogenase genes are important contributors to atmospheric hydrogen (H2 ) uptake in soil environments. Although previous studies reported the occurrence of a significant H2 uptake activity in vegetation, there has been no report on the identification and diversity of the responsible microorganisms. Here, we show the existence of plant-associated bacteria with the ability to consume atmospheric H2 that may be a potential energy source required for their persistence in plants. Detection of the gene hhyL - encoding the large subunit of group 5 [NiFe]-hydrogenase - in plant tissues showed that plant-associated high-affinity H2 -oxidizing bacteria are widely distributed in herbaceous plants. Among a collection of 145 endophytic isolates, 7 Streptomyces strains were shown to possess hhyL gene and exhibit high- or intermediate-affinity H2 uptake activity. Inoculation of Arabidopsis thaliana (thale cress) and Oryza sativa (rice) seedlings with selected isolates resulted in an internalization of the bacteria in plant tissues. H2 uptake activity per bacterial cells was comparable between plant and soil, demonstrating that both environments are favorable for the H2 uptake activity of streptomycetes. This study first demonstrated the occurrence of plant-associated high-affinity H2 -oxidizing bacteria and proposed their potential contribution as a sink for atmospheric H2 .

The syntrophic degradation of branched-chain fatty acids (BCFAs) such as 2-methylbutyrate and isobutyrate is an essential step in the production of methane from proteins/amino acids in anaerobic ecosystems. While a few syntrophic BCFA-degrading bacteria have been isolated, their metabolic pathways in BCFA and short-chain fatty acid (SCFA) degradation as well as energy conservation systems remain unclear. In an attempt to identify these pathways, we herein performed comparative genomics of three syntrophic bacteria: 2-methylbutyrate-degrading "Syntrophomonas wolfei subsp. methylbutyratica" strain JCM 14075(T) (=4J5(T)), isobutyrate-degrading Syntrophothermus lipocalidus strain TGB-C1(T), and non-BCFA-metabolizing S. wolfei subsp. wolfei strain Göttingen(T). We demonstrated that 4J5 and TGB-C1 both encode multiple genes/gene clusters involved in β-oxidation, as observed in the Göttingen genome, which has multiple copies of genes associated with butyrate degradation. The 4J5 genome possesses phylogenetically distinct β-oxidation genes, which may be involved in 2-methylbutyrate degradation. In addition, these Syntrophomonadaceae strains harbor various hydrogen/formate generation systems (i.e., electron-bifurcating hydrogenase, formate dehydrogenase, and membrane-bound hydrogenase) and energy-conserving electron transport systems, including electron transfer flavoprotein (ETF)-linked acyl-CoA dehydrogenase, ETF-linked iron-sulfur binding reductase, ETF dehydrogenase (FixABCX), and flavin oxidoreductase-heterodisulfide reductase (Flox-Hdr). Unexpectedly, the TGB-C1 genome encodes a nitrogenase complex, which may function as an alternative H2 generation mechanism. These results suggest that the BCFA-degrading syntrophic strains 4J5 and TGB-C1 possess specific β-oxidation-related enzymes for BCFA oxidation as well as appropriate energy conservation systems to perform thermodynamically unfavorable syntrophic metabolism.

Under methanogenic conditions, short-chain fatty acids are common byproducts from degradation of organic compounds and conversion of these acids is an important component of the global carbon cycle. Due to the thermodynamic difficulty of propionate degradation, this process requires syntrophic interaction between a bacterium and partner methanogen; however, the metabolic strategies and behavior involved are not fully understood. In this study, the first genome analysis of obligately syntrophic propionate degraders (Pelotomaculum schinkii HH and P. propionicicum MGP) and comparison with other syntrophic propionate degrader genomes elucidated novel components of energy metabolism behind Pelotomaculum propionate oxidation. Combined with transcriptomic examination of P. schinkii behavior in co-culture with Methanospirillum hungatei, we found that formate may be the preferred electron carrier for P. schinkii syntrophy. Propionate-derived menaquinol may be primarily re-oxidized to formate, and energy was conserved during formate generation through newly proposed proton-pumping formate extrusion. P. schinkii did not overexpress conventional energy metabolism associated with a model syntrophic propionate degrader Syntrophobacter fumaroxidans MPOB (i.e., CoA transferase, Fix, and Rnf). We also found that P. schinkii and the partner methanogen may also interact through flagellar contact and amino acid and fructose exchange. These findings provide new understanding of syntrophic energy acquisition and interactions. This article is protected by copyright. All rights reserved., We thank Steven Aalvink for scanning electron microscopy analysis and WEMC for making the system available. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement n. [323009] and a Gravitation Grant (Project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Organisation for Scientific Research (NWO). This work was also supported by The Japan Society for the Promotion of Science with Grant-in-Aid for Scientific Research No. 18H03367 to MK Nobu and 17H05239 and 18H01576 to T Narihiro., info:eu-repo/semantics/publishedVersion

Most microorganisms living in the environment have yet to be cultured, owing at least in part to their slow and poor propagation properties and susceptibility to oxidative stress. Our previous studies demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving the phosphate and agar separately (termed “PS” medium), can greatly improve the culturability of microorganisms by mitigating oxidative stress compared with the use of “PT” medium (autoclaving the phosphate and agar together). Here, we attempted to isolate phylogenetically novel bacteria by combining PS medium with prolonged cultivation. After inoculation with forest soil or pond sediment samples, significantly more colonies appeared on PS medium than on PT medium. A total of 98 and 74 colonies that emerged after more than 7 days of cultivation were isolated as slow growers from PS and PT media, respectively. Sequencing analysis of their 16S rRNA genes revealed that the slow growers recovered from PS medium included more phylogenetically novel bacteria than those from PT medium, including a strain that could be classified into a novel order in the class Alphaproteobacteria. Further physiological analysis of representative strains showed that they were actually slow and poor growers and formed small but visible colonies only on PS medium. This study demonstrates that the culturability of previously uncultured bacteria can be improved by using an isolation strategy that combines a simple modification in medium preparation with an extended incubation time. IMPORTANCE Most microbial species inhabiting natural environments have not yet been isolated. One of the serious issues preventing their isolation is intrinsically slow and/or poor growth. Moreover, these slow and/or poor growers are likely to be highly sensitive to environmental stresses, especially oxidative stress. We reported previously that interaction between agar and phosphate during autoclave sterilization generates hydrogen peroxide, which adversely affects the culturability of environmental microorganisms, in particular, slow-growing organisms vulnerable to oxidative stress. In this study, we successfully isolated many slow-growing bacterial strains with phylogenetic novelty by simply modifying their cultivation on agar plates, i.e., autoclaving the phosphate and agar separately. The current limited repertoire of culture techniques still has room for improvement in the isolation of microorganisms previously considered unculturable.

A Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacterium, designated strain RPE64T, was isolated from the gut symbiotic organ of the bean bug Riptortus pedestris, collected in Tsukuba, Japan, in 2007. 16S rRNA gene sequencing showed that this strain belongs to the Burkholderia glathei clade, exhibiting the highest sequence similarity to Burkholderia peredens LMG 29314T (100 %), Burkholderia turbans LMG 29316T (99.52 %) and Burkholderia ptereochthonis LMG 29326T (99.04 %). Phylogenomic analyses based on 107 single-copy core genes and Genome blast Distance Phylogeny confirmed B. peredens LMG 29314T , B. ptereochthonis LMG 29326T and several uncultivated, endophytic Burkholderia species as its nearest phylogenetic neighbours. Digital DNA–DNA hybridization experiments unambiguously demonstrated that strain RPE64T represents a novel species in this lineage. The G+C content of its genome was 63.2 mol%. The isoprenoid quinone was ubiquinone 8 and the predominant fatty acid components were C16 : 0, C18 : 1 ω7c and C17 : 0 cyclo. The absence of nitrate reduction and the capacity to grow at pH 8 clearly differentiated strain RPE64T from related Burkholderia species. Based on these genotypic and phenotypic characteristics, strain RPE64T is classified as representing a novel species of the genus Burkholderia , for which the name Burkholderia insecticola sp. nov. is proposed. The type strain is RPE64T (=NCIMB 15023T=JCM 31142T).

A moderately thermophilic, aerobic, stalked bacterium (strain MA2T) was isolated from marine sediments in Kagoshima Bay, Japan. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain MA2T was most closely related to the genera Rhodobium , Parvibaculum , and Rhodoligotrophos (92–93 % similarity) within the class Alphaproteobacteria . Strain MA2T was a Gram-stain-negative and stalked dimorphic bacteria. The temperature range for growth was 16–48 °C (optimum growth at 42 °C). This strain required yeast extract and NaCl (>1 %, w/v) for growth, tolerated up to 11 % (w/v) NaCl, and was capable of utilizing various carbon sources. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The DNA G+C content was 60.7 mol%. Strain MA2T performed denitrification and produced N2O from nitrate under strictly microaerobic conditions. Strain MA2T possessed periplasmic nitrate reductase (Nap) genes but not membrane-bound nitrate reductase (Nar) genes. On the basis of this morphological, physiological, biochemical and genetic information a novel genus and species, Tepidicaulis marinus gen. nov., sp. nov., are proposed, with MA2T ( = NBRC 109643T = DSM 27167T) as the type strain of the species.

Our previous study shows that an anaerobic intestinal bacterium strain AJ110941P contributes to type 2 diabetes development in mice. Here we phylogenetically and physiologically characterized this unique mouse gut bacterium. The 16S rRNA gene analysis revealed that the strain belongs to the family Lachnospiraceae but shows low sequence similarities ( P also matched with mouse gut-derived datasets (56% of total datasets) with > 99% similarity, suggesting that AJ110941P-related bacteria mainly reside in mouse digestive tracts. Strain AJ110941P shared common physiological traits (e.g., Gram-positive, anaerobic, mesophilic, and fermentative growth with carbohydrates) with relative species of the Lachnospiraceae. Notably, the biofilm-forming capacity was found in both AJ110941P and relative species. However, AJ110941P possessed far more strong ability to produce biofilm than relative species and formed unique structure of extracellular polymeric substances. Furthermore, AJ110941P cells are markedly long fusiform-shaped rods (9.0–62.5 µm) with multiple flagella that have never been observed in any other Lachnospiraceae members. Based on the phenotypic and phylogenetic features, we propose a new genus and species, Fusimonas intestini gen. nov., sp. nov. for strain AJ110941P (FERM BP-11443).

A novel thermophilic, anaerobic, chemoheterotrophic, acetate-oxidizing and iron(III)-, manganese(IV)-, nitrate- and sulfate-reducing bacterium, designated strain ANAT, was isolated from a deep subsurface oil field in Japan (Yabase oil field, Akita Pref.). Cells of strain ANAT were Gram-stain-negative, non-motile, non-spore forming and slightly curved or twisted rods (1.5–5.0 µm long and 0.6–0.7 µm wide). The isolate grew at 25–60 °C (optimum 55 °C) and pH 6.0–8.0 (optimum pH 7.0). The isolate was capable of reducing iron(III), manganese(IV), nitrate and sulfate as an electron acceptor. The isolate utilized a limited range of electron donors such as acetate, lactate, pyruvate and yeast extract for iron reduction. Strain ANAT also used pyruvate, fumarate, succinate, malate, yeast extract and peptone for fermentative growth. The major respiratory quinones were menaquinone-7(H8) and menaquinone-8. The strain contained C18 : 0, iso-C18 : 0 and C16 : 0 as the major cellular fatty acids. The G+C content of the genomic DNA was 34.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain ANAT was closely related to Calditerrivibrio nitroreducens in the phylum Deferribacteres with low sequence similarities (89.5 %), and formed a distinct clade within the family Deferribacteraceae . In addition, the isolate is the first sulfate-reducing member of the phylum Deferribacteres . Based on phenotypic, chemotaxonomic and phylogenetic properties, a novel genus and species, Petrothermobacter organivorans gen. nov., sp. nov., is proposed for the isolate (type strain=ANAT= NBRC 112621T=DSM 105015T).

N -Acylhomoserine lactone acylase (AHL acylase) is a well-known enzyme responsible for disrupting cell-cell communication (quorum sensing) in bacteria. Here, we isolated and characterized a novel and unique AHL acylase (designated MacQ) from a multidrug-resistant bacterium, Acidovorax sp. strain MR-S7. The purified MacQ protein heterologously expressed in Escherichia coli degraded a wide variety of AHLs, ranging from C 6 to C 14 side chains with or without 3-oxo substitutions. We also observed that AHL-mediated virulence factor production in a plant pathogen, Pectobacterium carotovorum , was dramatically attenuated by coculture with MacQ-overexpressing Escherichia coli , whereas E. coli with an empty vector was unable to quench the pathogenicity, which strongly indicates that MacQ can act in vivo as a quorum-quenching enzyme and interfere with the quorum-sensing system in the pathogen. In addition, this enzyme was found to be capable of degrading a wide spectrum of β-lactams (penicillin G, ampicillin, amoxicillin, carbenicillin, cephalexin, and cefadroxil) by deacylation, clearly indicating that MacQ is a bifunctional enzyme that confers both quorum quenching and antibiotic resistance on strain MR-S7. MacQ has relatively low amino acid sequence identity to any of the known acylases ( IMPORTANCE N -Acylhomoserine lactones (AHLs) are well-known signal molecules for bacterial cell-cell communication (quorum sensing), and AHL acylase, which is able to degrade AHLs, has been recognized as a major target for quorum-sensing interference (quorum quenching) in pathogens. In this work, we succeeded in isolating a novel AHL acylase (MacQ) from a multidrug-resistant bacterium and demonstrated that the MacQ enzyme could confer multidrug resistance as well as quorum quenching on the host organism. Indeed, the purified MacQ protein was found to be bifunctional and capable of degrading not only various AHL derivatives but also multiple β-lactam antibiotics by deacylation activities. Although quorum quenching and antibiotic resistance have been recognized to be distinct biological functions, our findings clearly link the two functions by discovering the novel bifunctional enzyme and further providing the possibility that a hitherto-overlooked antibiotic resistance mechanism mediated by the quorum-quenching enzyme may exist in natural environments and perhaps in clinical settings.

Viruses are diverse and play significant ecological roles in marine ecosystems. However, our knowledge of genome-level diversity in viruses is biased toward those isolated from few culturable hosts. Here, we determined 1,352 nonredundant complete viral genomes from marine environments. Lifting the uncertainty that clouds short incomplete sequences, whole-genome-wide analysis suggests that these environmental genomes represent hundreds of putative novel viral genera. Predicted hosts include dominant groups of marine bacteria and archaea with no isolated viruses to date. Some of the viral genomes encode many functionally related enzymes, suggesting a strong selection pressure on these marine viruses to control cellular metabolisms by accumulating genes., Metagenomics has revealed the existence of numerous uncharacterized viral lineages, which are referred to as viral “dark matter.” However, our knowledge regarding viral genomes is biased toward culturable viruses. In this study, we analyzed 1,600 (1,352 nonredundant) complete double-stranded DNA viral genomes (10 to 211 kb) assembled from 52 marine viromes. Together with 244 previously reported uncultured viral genomes, a genome-wide comparison delineated 617 genus-level operational taxonomic units (OTUs) for these environmental viral genomes (EVGs). Of these, 600 OTUs contained no representatives from known viruses, thus putatively corresponding to novel viral genera. Predicted hosts of the EVGs included major groups of marine prokaryotes, such as marine group II Euryarchaeota and SAR86, from which no viruses have been isolated to date, as well as Flavobacteriaceae and SAR116. Our analysis indicates that marine cyanophages are already well represented in genome databases and that one of the EVGs likely represents a new cyanophage lineage. Several EVGs encode many enzymes that appear to function for an efficient utilization of iron-sulfur clusters or to enhance host survival. This suggests that there is a selection pressure on these marine viruses to accumulate genes for specific viral propagation strategies. Finally, we revealed that EVGs contribute to a 4-fold increase in the recruitment of photic-zone viromes compared with the use of current reference viral genomes. IMPORTANCE Viruses are diverse and play significant ecological roles in marine ecosystems. However, our knowledge of genome-level diversity in viruses is biased toward those isolated from few culturable hosts. Here, we determined 1,352 nonredundant complete viral genomes from marine environments. Lifting the uncertainty that clouds short incomplete sequences, whole-genome-wide analysis suggests that these environmental genomes represent hundreds of putative novel viral genera. Predicted hosts include dominant groups of marine bacteria and archaea with no isolated viruses to date. Some of the viral genomes encode many functionally related enzymes, suggesting a strong selection pressure on these marine viruses to control cellular metabolisms by accumulating genes.

© 2017 International Society for Microbial Ecology All rights reserved. Members of the order Methanomicrobiales are abundant, and sometimes dominant, hydrogenotrophic (H2-CO2utilizing) methanoarchaea in a broad range of anoxic habitats. Despite their key roles in greenhouse gas emissions and waste conversion to methane, little is known about the physiological and genomic bases for their widespread distribution and abundance. In this study, we compared the genomes of nine diverse Methanomicrobiales strains, examined their pangenomes, reconstructed gene flow and identified genes putatively mediating their success across different habitats. Most strains slowly increased gene content whereas one, Methanocorpusculum labreanum, evidenced genome downsizing. Peat-dwelling Methanomicrobiales showed adaptations centered on improved transport of scarce inorganic nutrients and likely use H + rather than Na + transmembrane chemiosmotic gradients during energy conservation. In contrast, other Methanomicrobiales show the potential to concurrently use Na + and H + chemiosmotic gradients. Analyses also revealed that the Methanomicrobiales lack a canonical electron bifurcation system (MvhABGD) known to produce low potential electrons in other orders of hydrogenotrophic methanogens. Additional putative differences in anabolic metabolism suggest that the dynamics of interspecies electron transfer from Methanomicrobiales syntrophic partners can also differ considerably. Altogether, these findings suggest profound differences in electron trafficking in the Methanomicrobiales compared with other hydrogenotrophs, and warrant further functional evaluations.

A moderately thermophilic, methanol-oxidizing bacterium (strain Gela4T) was isolated from methane-utilizing mixed-culture originating from marine sediment near a hydrothermal vent. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain Gela4T was closely related to members of the genus ‘Methyloligella’ (94.7 % similarity) within the class Alphaproteobacteria . Strain Gela4T was a Gram‐staining‐negative and aerobic organism. Cells were rod-shaped and non-motile. The temperature range for growth of strain Gela4T was 19–43 °C (optimal growth at 35 °C). Strain Gela4T tolerated up to 9 % NaCl with an optimum at 1 %. The organism was a facultative methylotroph that could utilize methanol, methylamine, trimethylamine and a variety of multi-carbon compounds. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The predominant phospholipids were phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 63.9 mol%. On the basis of the morphological, physiological, biochemical and genetic information, a novel genus and species, Methyloceanibacter caenitepidi is proposed, with Gela4T ( = NBRC 109540T = DSM 27242T) as the type strain.

To date, the only methanoarchaea isolated directly from methane hydrate bearing sediments were Methanoculleus submarinus Nankai-1T and Methanoculleus sp. MH98A. Here, we provide the genome of Methanoculleus taiwanensis CYW4T isolated from the deep-sea subseafloor sediment at the Deformation Front offshore southwestern Taiwan, where methane hydrate deposits are likely located. Through comparative genomics analyses of nine Methanoculleus strains from various habitats, 2–3 coding genes for trehalose synthases were found in all nine Methanoculleus genomes, which were not detected in other methanogens and are therefore suggested as a signature of genus Methanoculleus among methane-producing archaea. In addition, the structural genes adjacent to trehalose synthase genes are comprised of the signaling module of Per-Arnt-Sim (PAS) domain-containing proteins, Hsp20 family proteins, arabinose efflux permeases and multiple surface proteins with fasciclin-like (FAS) repeat. This indicates that trehalose synthase gene clusters in Methanoculleus might play roles in the response to various stresses and regulate carbon storage and modification of surface proteins through accumulation of trehalose. The non-gas hydrate-associated Methanoculleus strains harbor carbon-monoxide dehydrogenase (cooS/acsA) genes, which are important for the conversion of acetate to methane at the step of CO oxidation/CO2 reduction in acetoclastic methanogens and further implies that these strains may be able to utilize CO for methanogenesis in their natural habitats. In addition, both genomes of M. bourgensis strains MS2T and MAB1 harbor highly abundant transposase genes, which may be disseminated from microbial communities in their habitats, sewage treatment plants and biogas reactors, which are breeding grounds for antibiotic resistance. Through comparative genomic analyses, we gained insight into understanding the life of strictly anaerobic methane-producing archaea in various habitats, especially in methane-based deep-sea ecosystems.

A low-diversity microbial community, dominated by the γ-proteobacterium Halomonas sulfidaeris, was detected in samples of warm saline formation porewater collected from the Cambrian Mt. Simon Sandstone in the Illinois Basin of the North American Midcontinent (1.8 km/5872 ft burial depth, 50°C, pH 8, 181 bars pressure). These highly porous and permeable quartz arenite sandstones are directly analogous to reservoirs around the world targeted for large-scale hydrocarbon extraction, as well as subsurface gas and carbon storage. A new downhole low-contamination subsurface sampling probe was used to collect in situ formation water samples for microbial environmental metagenomic analyses. Multiple lines of evidence suggest that this H. sulfidaeris-dominated subsurface microbial community is indigenous and not derived from drilling mud microbial contamination. Data to support this includes V1-V3 pyrosequencing of formation water and drilling mud, as well as comparison with previously published microbial analyses of drilling muds in other sites. Metabolic pathway reconstruction, constrained by the geology, geochemistry and present-day environmental conditions of the Mt. Simon Sandstone, implies that H. sulfidaeris-dominated subsurface microbial community may utilize iron and nitrogen metabolisms and extensively recycle indigenous nutrients and substrates. The presence of aromatic compound metabolic pathways suggests this microbial community can readily adapt to and survive subsurface hydrocarbon migration.

Despite their importance as a biofuel production platform, only a very limited number of butanol-tolerant bacteria have been identified thus far. Here, we extensively explored butanol- and isobutanol-tolerant bacteria from various environmental samples. A total of 16 aerobic and anaerobic bacteria that could tolerate greater than 2.0% (vol/vol) butanol and isobutanol were isolated. A 16S rRNA gene sequencing analysis revealed that the isolates were phylogenetically distributed over at least nine genera: Bacillus , Lysinibacillus , Rummeliibacillus , Brevibacillus , Coprothermobacter , Caloribacterium , Enterococcus , Hydrogenoanaerobacterium , and Cellulosimicrobium , within the phyla Firmicutes and Actinobacteria . Ten of the isolates were phylogenetically distinct from previously identified butanol-tolerant bacteria. Two relatively highly butanol-tolerant strains CM4A (aerobe) and GK12 (obligate anaerobe) were characterized further. Both strains changed their membrane fatty acid composition in response to butanol exposure, i.e., CM4A and GK12 exhibited increased saturated and cyclopropane fatty acids (CFAs) and long-chain fatty acids, respectively, which may serve to maintain membrane fluidity. The gene ( cfa ) encoding CFA synthase was cloned from strain CM4A and expressed in Escherichia coli . The recombinant E. coli showed relatively higher butanol and isobutanol tolerance than E. coli without the cfa gene, suggesting that cfa can confer solvent tolerance. The exposure of strain GK12 to butanol by consecutive passages even enhanced the growth rate, indicating that yet-unknown mechanisms may also contribute to solvent tolerance. Taken together, the results demonstrate that a wide variety of butanol- and isobutanol-tolerant bacteria that can grow in 2.0% butanol exist in the environment and have various strategies to maintain structural integrity against detrimental solvents.

Pantothenate synthetase (PanC) and pantothenate kinase which function in the canonical coenzyme A (CoA) biosynthetic pathway cannot be found in most archaea. COG1829 and COG1701 intrinsic to archaea were proposed as the candidate proteins for producing 4'-phosphopantothenate instead, and the COG1701 protein from Methanosarcina mazei was assigned as PanC. Meanwhile, the Thermococcus kodakarensis COG1829 and COG1701 proteins were biochemically identified as novel enzymes, i.e., pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). In this study, the functions of Mhun_0831 (COG1829) and Mhun_0832 (COG1701) from Methanospirillum hungatei were identified, and the recombinant enzymes were partially characterized. Plasmids simultaneously possessing the two genes encoding Mhun_0831 and Mhun_0832 complemented the poor growth of the temperature-sensitive Escherichia coli pantothenate kinase mutant ts9. The recombinant Mhun_0831 and Mhun_0832 expressed in E. coli cells exhibited PoK and PPS activities, respectively, being in accord with the functions of T. kodakarensis proteins. The PoK activity was most active at pH 8.5 and 40°C, and accepted ATP and UTP as a phosphate donor. Although CoA did not affect the PoK activity, the end product considerably accelerated the PPS activity. The homologs of both proteins are widely conserved in most archaeal genomes. Taken together, our findings indicate that archaea can synthesize CoA through the unique pathway involving PoK and PPS, in addition to the canonical one that the order Thermoplasmatales employs.

A thermophilic and hydrogenotrophic methanogen, strain RMAST, was isolated from gas-associated formation water of a gas-producing well in a natural gas field in Japan. Strain RMAST grew solely on H2/CO2 but required Casamino acids, tryptone, yeast extract or vitamins for growth. Growth of strain RMAST was stimulated by acetate. Cells were non-motile, straight rods (0.5×3.5–10.5 µm) and occurred singly or in pairs. Bundles of fimbriae occurred at both poles of cells and the cell wall was thick (approximately 21 nm, as revealed by ultrathin section electron microscopy). Strain RMAST grew at 45–80 °C (optimum, 70 °C), at pH 5.8–8.7 (optimum, pH 6.9–7.7) and with 0.001–20 g NaCl l−1 (optimum, 2.5 g NaCl l−1). Phylogenetic analysis revealed that Methanothermobacter thermautotrophicus ΔHT was most closely related to the isolate (95.7 % 16S rRNA gene sequence similarity). On the basis of morphological, phenotypic and phylogenetic characteristics, it is clear that strain RMAST represents a novel species of the genus Methanothermobacter , for which we propose the name Methanothermobacter tenebrarum sp. nov. The type strain is RMAST ( = DSM 23052T = JCM 16532T = NBRC 106236T).

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 B10(T) 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 B10(T), 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.

A free-living ciliate, Trimyema compressum , found in anoxic freshwater environments harbors methanogenic archaea and a bacterial symbiont named TC1 in its cytoplasm. Here, we report the complete genome sequence of the TC1 symbiont, consisting of a 1.59-Mb chromosome and a 35.8-kb plasmid, which was determined using the PacBio RSII sequencer.

C. difficile is a major enteric pathogen with worldwide distribution. Its expansion is associated with broad-spectrum antibiotics which disturb the normal gut microbiome. In this study, the DNA sequencing of highly active bacteria and bacteria opsonized by intestinal secretory immunoglobulin A (SIgA) separated from the whole bacterial community by FACS elucidated how the gut dysbiosis promotes C. difficile infection (CDI). Bacterial groups with inhibitory effects on C. difficile growth, such as Lactobacillales, were mostly inactive in the CDI patients. C. difficile was typical for the bacterial fraction opsonized by SIgA in patients with CDI, while Fusobacterium was characteristic for the SIgA-opsonized fraction of the controls. The study demonstrates that sequencing of specific bacterial fractions provides additional information about dysbiotic processes in the gut. The detected patterns have been confirmed with the whole patient cohort independently of the taxonomic differences detected in the nonfractionated microbiomes., The onset of Clostridium difficile infection (CDI) has been associated with treatment with wide-spectrum antibiotics. Antibiotic treatment alters the activity of gut commensals and may result in modified patterns of immune responses to pathogens. To study these mechanisms during CDI, we separated bacteria with high cellular RNA content (the active bacteria) and their inactive counterparts by fluorescence-activated cell sorting (FACS) of the fecal bacterial suspension. The gut dysbiosis due to the antibiotic treatment may result in modification of immune recognition of intestinal bacteria. The immune recognition patterns were assessed by FACS of bacterial fractions either coated or not with intestinal secretory immunoglobulin A (SIgA). We described the taxonomic distributions of these four bacterial fractions (active versus inactive and SIgA coated versus non-SIgA coated) by massive 16S rRNA gene amplicon sequencing and quantified the proportion of C. difficile toxin genes in the samples. The overall gut microbiome composition was more robustly influenced by antibiotics than by the C. difficile toxins. Bayesian networks revealed that the C. difficile cluster was preferentially SIgA coated during CDI. In contrast, in the CDI-negative group Fusobacterium was the characteristic genus of the SIgA-opsonized fraction. Lactobacillales and Clostridium cluster IV were mostly inactive in CDI-positive patients. In conclusion, although the proportion of C. difficile in the gut is very low, it is able to initiate infection during the gut dysbiosis caused by environmental stress (antibiotic treatment) as a consequence of decreased activity of the protective bacteria. IMPORTANCE C. difficile is a major enteric pathogen with worldwide distribution. Its expansion is associated with broad-spectrum antibiotics which disturb the normal gut microbiome. In this study, the DNA sequencing of highly active bacteria and bacteria opsonized by intestinal secretory immunoglobulin A (SIgA) separated from the whole bacterial community by FACS elucidated how the gut dysbiosis promotes C. difficile infection (CDI). Bacterial groups with inhibitory effects on C. difficile growth, such as Lactobacillales, were mostly inactive in the CDI patients. C. difficile was typical for the bacterial fraction opsonized by SIgA in patients with CDI, while Fusobacterium was characteristic for the SIgA-opsonized fraction of the controls. The study demonstrates that sequencing of specific bacterial fractions provides additional information about dysbiotic processes in the gut. The detected patterns have been confirmed with the whole patient cohort independently of the taxonomic differences detected in the nonfractionated microbiomes.

The family Methanomicrobiaceae comprises hydrogen- and formate-utilizing methanogens. Genome sequencing of nine species of Methanomicrobiaceae has been conducted so far. Here, we report three additional draft genome sequences of Methanomicrobiaceae , those of Methanoculleus horonobensis JCM 15517 (=T10 T ), Methanoculleus thermophilus DSM 2373 (=CR-1 T ), and Methanofollis ethanolicus JCM 15103 (=HASU T ).

Syntrophomonas wolfei subsp. methylbutyratica strain 4J5 T (=JCM 14075 T ) is a mesophilic bacterium capable of degrading butyrate and 2-methylbutyrate through syntrophic cooperation with a partner methanogen. The draft genome sequence is 3.2 Mb, with a G+C content of 45.5%.

Some 25 fungi, including at least 14 basidiomycetes, one ascomycete, and five anamorphic fungi were evaluated for their cellulose-degrading abilities in Difco potato dextrose broth or Difco malt extract broth cultures with cellulosic substrates (e.g., filter paper) in plastic Petri dishes. Among them, Peniophora sp. 06-13 and Phlebia sp. 99-335 reduced the dry weights of the whole cultures with these substrates more than the dry weights of the respective original substrates after 30 days of culture, showing definite cellulose degradation. In the cultures with more than 10 test fungi including Pycnoporus coccineus 84-117, such weight losses did not occur. This assay technique for the primary screening for cellulose degrading fungi is simple, inexpensive, reproducible and accurate.