Lutimonas zeaxanthinifaciens sp. nov., a zeaxanthin-producing marine bacterium isolated from coastal sediment.

A Gram-stain-negative, yellow-pigmented, strictly aerobic, non-flagellated, motile by gliding, rod-shaped bacterium, designated strain YSD2104T, was isolated from a coastal sediment sample collected from the southeastern part of the Yellow Sea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain YSD2104T was closely related to three type strains, Lutimonas vermicola IMCC1616T (97.4 %), Lutimonas saemankumensis SMK-142T (96.9 %), and Lutimonas halocynthiae RSS3-C1T (96.8 %). Strain YSD2104T has a single circular chromosome of 3.54 Mbp with a DNA G+C content of 38.3 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain YSD2104T and the three type strains (L. vermicola IMCC1616 T, L. saemankumensis SMK-142T, and L. halocynthiae RSS3-C1T) were 74.0, 86.2 and 73.6 %, and 17.9, 30.3 and 17.8 %, respectively. Growth was observed at 20-30 °C (optimum, 30 °C), at pH 6.5-8.5 (optimum, pH 7.0), and with NaCl concentrations of 1.5-3.5 % (optimum, 2.5 %). The major carotenoid was zeaxanthin, and flexirubin-type pigment was not produced. The major respiratory quinone was menaquinone-6. The major fatty acids (>10 %) were iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), and summed feature 9 (iso-C17 : 1 ω9c and/or 10-methyl C16 : 0). The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, two unidentified aminolipids, and eight unidentified lipids. Conclusively, based on this polyphasic approach, we classified strain YSD2104T (=KCTC 102008T=JCM 36287T) as representing a novel species of the genus Lutimonas and proposed the name Lutimonas zeaxanthinifaciens sp. nov.

Synthesis of Silver Nanoparticles Using Aggregatimonas sangjinii F202Z8T and Their Biological Characterization.

The aim of this study is to describe the general features and eco-friendly biosynthesis of silver nanoparticles (AgNPs) from the marine bacterium Aggregatimonas sangjinii F202Z8T. To the best of our knowledge, no previous study has reported the biosynthesis of AgNPs using this strain. The formation of AgNPs using F202Z8T was synthesized intracellularly without the addition of any disturbing factors, such as antibiotics, nutrient stress, or electron donors. The AgNPs were examined using UV-vis spectrophotometry, transmission electron microscopy, energy-dispersive X-ray spectroscopy, nanoparticle tracking analysis, and Fourier transform infrared spectrometry. The UV-vis spectrum showed a peak for the synthesized AgNPs at 465 nm. The AgNPs were spherical, with sizes ranging from 27 to 82 nm, as denoted by TEM and NTA. FTIR showed various biomolecules including proteins and enzymes that may be involved in the synthesis and stabilization of AgNPs. Notably, the AgNPs demonstrated broad-spectrum antibacterial effects against various pathogenic Gram-positive and Gram-negative bacteria, including Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. The minimum inhibitory concentrations and minimum bactericidal concentrations of the F202Z8T-formed AgNPs were 80 and 100 µg/mL, 40 and 50 µg/mL, and 30 and 40 µg/mL against E. coli, B. subtilis, and S. aureus, respectively. This study suggests that A. sangjinii F202Z8T is a candidate for the efficient synthesis of AgNPs and may be suitable for the formulation of new types of bactericidal substances.

Falsirhodobacter algicola sp. nov., a member of the Rhodobacteraceae isolated from the marine red algae Grateloupia sp.

A Gram-negative, pale yellow-pigmented, non-flagellated, motile, rod-shaped and aerobic bacterium, designated strain PG104T, was isolated from red algae Grateloupia sp. collected from the coastal area of Pohang, Republic of Korea. Growth of strain PG104T was observed at 15-35 °C (optimum, 30 °C), pH 6.0-10.0 (optimum, pH 7.5-8.0) and in the presence of 0-8.0 % (w/v) NaCl (optimum, 5.0 %). The predominant fatty acids included C17 : 0, C18 : 0, 11-methyl C18 : 1 ω7c and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and the major respiratory quinone was Q-10. Polar lipids included phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified lipid and one unidentified aminolipid. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain PG104T formed a phylogenetic lineage with members of the genus Falsirhodobacter and exhibited 16S rRNA gene sequence similarities of 97.1 and 96.6 % to Falsirhodobacter deserti W402T and Falsirhodobacter halotolerans JA744T, respectively. The complete genome of strain PG104T consisted of a single circular chromosome of approximately 2.8 Mbp with five plasmids. Based on polyphasic taxonomic data, strain PG104T represents a novel species in the genus Falsirhodobacter, for which the name Falsirhodobacter algicola sp. nov. is proposed. The type strain of Falsirhodobacter algicola is PG104T (=KCTC 82230T=JCM 34380T).

In vitro and in vivo antimicrobial activity of the fungal metabolite toluquinol against phytopathogenic bacteria.

Introduction: Bacterial plant diseases cause tremendous economic losses worldwide. However, a few effective and sustainable control methods are currently available. To discover novel and effective management approaches, we screened marine fungi for their antibacterial activity against phytopathogenic bacteria in vitro and in vivo. Methods: We screened the culture broth of 55 fungal strains isolated from various marine sources (seawater, algae, and sediment) for their in vitro antibacterial activity using the broth microdilution method. Then, only the fungal strain (designated UL-Ce9) with higher antibacterial activity in vitro was tested in an in vivo experiment against tomato bacterial wilt. The active compounds of UL-Ce9 were extracted using ethyl acetate, purified by a series of chromatography, and the structure was elucidated by nuclear magnetic resonance spectroscopy. Pesticide formulations of toluquinol were prepared as soluble concentrates and wettable powder. The disease control efficacy of toluquinol formulations was evaluated against blight of rice and the bacterial wilt of tomato. Results and discussion: The culture broth of UL-Ce9 showed high antibacterial activity against Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas arboricola pv. pruni in vitro, and we selected UL-Ce9 for the in vivo test. The UL-Ce9 culture broth completely suppressed the bacterial wilt of tomato at a dilution of 1:5. The phylogenetic analysis identified UL-Ce9 as Penicillium griseofulvum, and the antibacterial metabolites were revealed as patulin, gentisyl alcohol, and toluquinol, all of which were associated with the biosynthetic pathway of the mycotoxin patulin. Patulin exhibited the highest antibacterial activity against 16 phytopathogenic bacteria in vitro, followed by toluquinol and gentisyl alcohol. As patulin is toxic, we selected toluquinol to investigate its potential use as a pesticide against bacterial plant diseases. Compared with the chemicals currently being applied in agriculture (streptomycin and oxytetracycline), toluquinol formulations exhibited similar and higher control efficacies against bacterial leaf blight of rice and bacterial wilt of tomato, respectively. To the best of our knowledge, this is the first report of the antibacterial activity of toluquinol against phytopathogenic bacteria. Our results suggest that toluquinol is a potential candidate for the development of novel and effective pesticides for the management of bacterial plant diseases.

Fulvivirga lutea sp. nov., a marine bacterium isolated from seawater.

A strictly aerobic, Gram-stain-negative, gliding, rod-shaped bacteria, designated strain S481T, was isolated from a surface seawater sample collected at Gunsan marina, in the West Sea of the Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain S481T formed a monophyletic clade with members of the genus Fulvivirga, showing 93.7-95.8% sequence similarity to the type strains. Strain S481T has a single circular chromosome of 4.13 Mbp with a DNA G+C content of 37.3 mol%. The values of average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization between strain S481T and all genome-sequenced species of the genus Fulvivirga were below 71.2%, 68.6% and 18.9%, respectively, indicating lower values than the standard cut-offs for species delineation. Growth was observed at 20-42 °C (optimum, 37 °C), at pH 6-8 (optimum, pH 7) and with 0 - 6 % NaCl (optimum, 1-2 %). The major fatty acids (>10%) were iso-C15:0, iso-C15:1 G and C16:1ω5c. The respiratory quinone was MK-7. The major polar lipids were identified as phosphatidylethanolamine, three unidentified aminolipids and five unidentified lipids. Based on the results of phenotypic characterization, phylogenetic analysis and genome-based comparison, strain S481T represents a novel species in the genus Fulvivirga, for which we propose the name Fulvivirga lutea sp. nov. The type strain is S481T (=KCTC 82209T=JCM 34505T).

Retinaldehyde Dehydrogenase Inhibition-Related Adverse Outcome Pathway: Potential Risk of Retinoic Acid Synthesis Inhibition during Embryogenesis.

Retinoic acid (RA) is one of the factors crucial for cell growth, differentiation, and embryogenesis; it interacts with the retinoic acid receptor and retinoic acid X receptor to eventually regulate target gene expression in chordates. RA is transformed from retinaldehyde via oxidization by retinaldehyde dehydrogenase (RALDH), which belongs to the family of oxidoreductases. Several chemicals, including disulphiram, diethylaminobenzaldehyde, and SB-210661, can effectively inhibit RALDH activity, potentially causing reproductive and developmental toxicity. The modes of action can be sequentially explained based on the molecular initiating event toward key events, and finally the adverse outcomes. Adverse outcome pathway (AOP) is a conceptual and theoretical framework that describes the sequential chain of casually liked events at different biological levels from molecular events to adverse effects. In the present review, we discussed a recently registered AOP (AOP297; inhibition of retinaldehyde dehydrogenase leads to population decline) to explain and support the weight of evidence for RALDH inhibition-related developmental toxicity using the existing knowledge.

Ferrimonas lipolytica sp. nov., a facultatively anaerobic bacterium isolated from seawater.

A Gram-stain-negative, motile, facultatively anaerobic rod-shaped bacterium with a polar flagellum, designated strain S7T was isolated from seawater sample collected at Uljin marina, in the East Sea of the Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain S7T was affiliated with members of genus Ferrimonas, showing the highest sequence similarities to the type strains Ferrimonas senticii P2S11T (95.7 %), Ferrimonas balearica PATT (95.7 %) and Ferrimonas pelagia CBA4601T (95.1 %). The genome was 4.13 Mbp with a DNA G+C content of 49.4 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between S7T and F. senticii P2S11T and F. balearica PATT yielded ANI values of 71.9 and 70.7 %, and dDDH values of 15.1 and 13.9 %, respectively. The genome of S7T was predicted to encode triacylglycerol lipase, phospholipase A1/A2 and lysophospholipase as well as esterase involved in lipolytic processes. Growth was observed at 8-31 °C (optimum 27 °C), at pH 7-9 (optimum pH 7), and with 1-6 % NaCl (optimum 2 %). The respiratory quinones were MK-7 and Q-7 and the major fatty acids (>10 %) were C16 : 0, C16 : 1ω9c, C17 : 1ω8c, and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The major polar lipids were identified as phosphatidylethanolamine, phosphatidylglycerol, two unidentified phospholipids, and three unidentified lipids. On the basis of the results of this polyphasic analysis, it was determined that the strain represents a novel species of the genus Ferrimonas, for which the name Ferrimonas lipolytica sp. nov. is proposed. The type strain is S7T (=KCTC 72490T=JCM 33793T).

Shewanella maritima sp. nov., a facultative anaerobic marine bacterium isolated from seawater, and emended description of Shewanella intestini.

A Gram-stain-negative, motile, facultative anaerobic rod-shaped marine bacterium, designated strain D4-2T, was isolated from a sample of seawater collected at Dong-do marina, Dokdo Island, in the East Sea of the Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain D4-2T was affiliated with members of genus Shewanella and closely related to Shewanella intestini XMDDZSB0408T (97.4%), followed by Shewanella gelidii RZB5-4T (96.7 %) and Shewanella inventionis KX27T (96.1 %). D4-2T has a single circular chromosome of 4.72 Mbp with a DNA G+C content of 44.5 mol%. Average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values between strain D4-2T and the previously mentioned type strains of species of the genus Shewanella were in range of 69-83.8 % and 20.5-21.7 %, respectively. Growth was observed at 10-36 °C (optimum 29-32 °C), at pH 6-9 (optimum pH 7), and with 1-6% NaCl (optimum 2%). The predominant fatty acids (>10 %) of D4-2T were iso-C15:0 and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The respiratory quinones were Q-7, Q-8, MK-7 and MMK-7. Phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminophospholipid, an unidentified aminolipid and four unidentified lipids were detected in D4-2T. On the basis of phenotypic, chemotaxonomic and molecular properties, D4-2T represents a novel species of the genus Shewanella, for which the name Shewanella maritima sp. nov. is proposed with D4-2T as the type strain (=KCTC 72040T=JCM 33294T).

Tamlana carrageenivorans sp. nov., a carrageenan-degrading bacterium isolated from seawater.

A Gram-stain-negative, aerobic, non-motile, rod-shaped, agarolytic and carrageenolytic bacterial strain, designated UJ94T, was isolated from seawater of Uljin in the Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain UJ94T shared sequence similarities of 98.4, 96.1 and 95.4 % with Tamlana agarivorans JW-26T, Tamlana sedimentorum KMM 9545T and Tamlana crocina HST1-43T, respectively. Growth of strain UJ94T was observed at 4-37 °C and pH 6.5-8.0 in the presence of 2-9 % (w/v) NaCl. The major fatty acids of strain UJ94T were iso-C15 : 0, summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and iso-C17 : 0 3-OH; MK-6 was the predominant menaquinone. Phosphatidylethanolamine, two unidentified aminolipids and five unidentified lipids were detected as major polar lipids. The whole circular genome comprised 4 116 543 bp and had a G+C content of 35.2 mol%. The ranges of average nucleotide identity and in silico DNA-DNA hybridization estimated by genome-to-genome distance were 90.6-74.2 % and 47.6-14.6 %, respectively, with the type strains of T. agarivorans and T. sedimentorum. The present polyphasic study, including phylogenetic, chemotaxonomic, biochemical and genomic data, suggested that strain UJ94T represents a novel species of the genus Tamlana, for which the name Tamlana carrageenivorans sp. nov. is proposed. The type strain is UJ94T (=KCTC 62451T=NBRC 113234T).

Identification and characterization of a marine-derived chitinolytic fungus, Acremonium sp. YS2-2.

Chitin is the most abundant biopolymer in marine environments. To facilitate its utilization, our laboratory screened marine-derived fungal strains for chitinolytic activity. One chitinolytic strain isolated from seawater, designated YS2-2, was identified as Acremonium species based on morphological and phylogenetic analyses. Acremonium species are cosmopolitan fungi commonly isolated from both terrestrial and marine environments, but their chitinolytic activity is largely unknown. The extracellular crude enzyme of YS2-2 exhibited optimum chitinolytic activity at pH 6.0-7.6, 23-45°C, and 1.5% (w/v) NaCl. Degenerate PCR revealed the partial cDNA sequence of a putative chitinase gene, chiA, in YS2-2. The expression of chiA was dramatically induced in response to 1% (w/v) colloidal chitin compared to levels under starvation, chitin powder, and glucose conditions. Moreover, the chiA transcript levels were positively correlated with chitinolytic activities under various colloidal chitin concentrations, suggesting that ChiA mediates chitinolytic activity in this strain. Our results provide a basis for additional studies of marinederived chitinolytic fungi aimed at improving industrial applications.

Complete Genome Sequence of Marinobacterium aestuarii ST58-10T, a Benzene-Degrading Bacterium Isolated from Estuarine Sediment.

Marinobacterium aestuarii ST58-10T was identified as a benzene-degrading aerobic bacterium isolated from estuarine sediment in the Republic of Korea. The genome of strain ST58-10T was found to be composed of a single circular chromosome (5,191,608 bp) with a G+C content of 58.78% and harboring 4,473 protein-coding genes. The assembled sequence data will help elucidate potential metabolic pathways and mechanisms responsible for the hydrocarbon-degrading ability of M. aestuarii ST58-10T.

Flavobacterium sediminis sp. nov., a starch-degrading bacterium isolated from tidal flat sediment.

A yellow-pigmented bacterium with the ability to degrade starch, designated MEBiC07310T, was isolated from tidal flat sediment collected in Taean County, Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain MEBiC07310T was affiliated with the genus Flavobacterium in the phylum Bacteroidetes and showed that the strain was most closely related to Flavobacterium haoranii LQY-7T (96.8 % similarity), followed by Flavobacterium indicum GPTSA 100-9T (95.2 %) and Flavobacterium urocaniciphilum YIT 12746T (94.6 %). Genome-based analysis of the average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) of strain MEBiC07310T compared with F. haoranii LQY-7T and F. indicum GPTSA 100-9T yielded ANI values of 77.0 and 73.3 % and DDH values of 18.0±2.7 and 16.1±3.6 %, respectively. The DNA G+C content of strain MEBiC07310T was 35.2 mol%. Cells of the strain were aerobic, Gram-stain-negative and rod-shaped, and negative for flexirubin-type pigments. Growth was observed at 17-43 °C (optimum 32 °C), at pH 5.0-8.0 (optimum pH 7.0) and with 0-3 % (w/v) NaCl (optimum 1 %). The major fatty acids (>10 %) of strain MEBiC07310T were iso-C15 : 0, iso-C17 : 0 3-OH, summed feature 1 (iso-C15 : 1 H and/or C13 : 0 3-OH) and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). The major respiratory quinone was menaquinone MK-6. Based on its phenotypic and genotypic characteristics, strain MEBiC07310T should be classified as representing a novel species of the genus Flavobacterium, for which the name Flavobacterium sediminis sp. nov. is proposed. The type strain is MEBiC07310T (=KCTC 62132T=JCM 32291T).

Marinobacterium aestuarii sp. nov., a benzene-degrading marine bacterium isolated from estuary sediment.

A Gram-stain-negative, aerobic, motile, flagellated rod-shaped bacterium, designated ST58-10T, was isolated from an estuarine sediment in the Republic of Korea. The strain was able to degrade benzene. Growth of strain ST58-10T was observed at 4-35 °C (optimum, 20-25 °C), pH 5-9 (optimum, pH 7-8) and 1-8 % NaCl (optimum, 3 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ST58-10T formed a phyletic lineage within the genus Marinobacterium of the family Oceanospirillaceae. Strain ST58-10T was most closely related to Marinobacterium profundum PAMC 27536T (99.6 %) and Marinobacterium rhizophilum CL-YJ9T (98.3 %), and to other members of the genus Marinobacterium(94.5-91.5 %). However, the mean value estimated by using the Genome-to-Genome Distance Calculator was 50.6±7.4 % with M. profundum PAMC 27536T and 30.9±2.8 with M. rhizophilum CL-YJ9T, respectively. An average nucleotide identity value was 89.0 % with M. profundum PAMC 27536T and 85.6 % with M. rhizophilum CL-YJ9T, respectively. The major fatty acids of strain ST58-10T were summed feature 3 (comprising C16 : 1ω7c/C16 : 1ω6c), summed feature 8 (comprising C18 : 1 ω7c/C18 : 1ω6c), C16 : 0 and C10 : 0 3-OH, and contained ubiquinone (Q-8) as the sole isoprenoid quinone. Phosphatidylethanolamine, phosphatidylglycerol, three unidentified aminolipids, an unidentified glycolipid and an unidentified lipid were detected as polar lipids. The DNA G+C content of strain ST58-10T was 58.78 mol%. On the basis of the phenotypic, chemotaxonomic and molecular properties, strain ST58-10T represents a novel species of the genus Marinobacterium, for which the name Marinobacterium aestuarii sp. nov. is proposed. The type strain is ST58-10T (=KCTC 52193T=NBRC 112103T).

Echinicola strongylocentroti sp. nov., isolated from a sea urchin Strongylocentrotus intermedius.

A yellowish-orange-pigmented marine bacterium, designated MEBiC08714T was isolated from a sea urchin Strongylocentrotus intermedius collected at the west edge of the East Sea of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that MEBiC08714T was affiliated with the genus Echinicola and that the strain was most closely related to Echinicola vietnamensis KCTC 12713T (96.9 %), followed by 'Echinicola shivajiensis' JCM 17847 (96.3 %), Echinicola jeungdonensis KCTC 23122T (96.1 %), and Echinicola pacifica KCTC 12368T (95.0 %). Cells were strictly aerobic, Gram-stain-negative, rod shaped, flexirubin-type pigments-negative and motile by gliding. Growth was observed at 20-35 °C (optimum 25 °C), at pH 6-11 (optimum pH 7.0), and with 0-13 % NaCl (optimum 2 %). This strain was able to hydrolyze agar and starch. The polar lipids of MEBiC08714T contained phosphatidylethanolamine, an unidentified phospholipid, and four unidentified lipids. The major fatty acids were iso-C15 : 0 (27.5 %), iso-C17 : 0 3-OH (11.5 %), anteiso-C15 : 0 (5.2 %), summed feature 3 (C16:1ω7c and/or C16:1ω6c, 20.3 %) and summed feature 9 (iso-C17 : 1ω9c and/or10-methyl C16 : 06, 6.3 %). The DNA G+C content was 44.2 mol% and the major respiratory quinone was MK-7. On the basis of these polyphasic taxonomic data, MEBiC08714T represents a novel species of the genus Echinicola, for which the name Echinicola strongylocentroti sp. nov. is proposed. The type strain is MEBiC08714T (=KCTC 52052T=JCM 31307T).

A biological process effective for the conversion of CO-containing industrial waste gas to acetate.

Acetogens have often been observed to be inhibited by CO above an inhibition threshold concentration. In this study, a two-stage culture consisting of carboxydotrophic archaea and homoacetogenic bacteria is found to be effective in converting industrial waste gas derived from a steel mill process. In the first stage, Thermococcus onnurineus could grow on the Linz-Donawitz converter gas (LDG) containing ca. 56% CO as a sole energy source, converting the CO into H2 and CO2. Then, in the second stage, Thermoanaerobacter kivui could grow on the off-gas from the first stage culture, consuming the H2 and CO in the off-gas completely and producing acetate as a main product. T. kivui alone could not grow on the LDG gas. This work represents the first demonstration of acetate production using steel mill waste gas by a two-stage culture of carboxydotrophic hydrogenogenic microbes and homoacetogenic bacteria.

Adaptive engineering of a hyperthermophilic archaeon on CO and discovering the underlying mechanism by multi-omics analysis.

The hyperthermophilic archaeon Thermococcus onnurineus NA1 can grow and produce H2 on carbon monoxide (CO) and its H2 production rates have been improved through metabolic engineering. In this study, we applied adaptive evolution to enhance H2 productivity. After over 150 serial transfers onto CO medium, cell density, CO consumption rate and H2 production rate increased. The underlying mechanism for those physiological changes could be explained by using multi-omics approaches including genomic, transcriptomic and epigenomic analyses. A putative transcriptional regulator was newly identified to regulate the expression levels of genes related to CO oxidation. Transcriptome analysis revealed significant changes in the transcript levels of genes belonging to the categories of transcription, translation and energy metabolism. Our study presents the first genome-scale methylation pattern of hyperthermophilic archaea. Adaptive evolution led to highly enhanced H2 productivity at high CO flow rates using synthesis gas produced from coal gasification.

Extracellular Vesicles of the Hyperthermophilic Archaeon "Thermococcus onnurineus" NA1T.

Extracellular vesicles (EVs) produced by a sulfur-reducing, hyperthermophilic archaeon, "Thermococcus onnurineus" NA1(T), were purified and characterized. A maximum of four EV bands, showing buoyant densities between 1.1899 and 1.2828 g cm(-3), were observed after CsCl ultracentrifugation. The two major EV bands, B (buoyant density at 25°C [ρ(25)] = 1.2434 g cm(-3)) and C (ρ(25) = 1.2648 g cm(-3)), were separately purified and counted using a qNano particle analyzer. These EVs, showing different buoyant densities, were identically spherical in shape, and their sizes varied from 80 to 210 nm in diameter, with 120- and 190-nm sizes predominant. The average size of DNA packaged into EVs was about 14 kb. The DNA of the EVs in band C was sequenced and assembled. Mapping of the T. onnurineus NA1(T) EV (ToEV) DNA sequences onto the reference genome of the parent archaeon revealed that most genes of T. onnurineus NA1(T) were packaged into EVs, except for an ∼9.4-kb region from TON_0536 to TON_0544. The absence of this specific region of the genome in the EVs was confirmed from band B of the same culture and from bands B and C purified from a different batch culture. The presence of the 3'-terminal sequence and the absence of the 5'-terminal sequence of TON_0536 were repeatedly confirmed. On the basis of these results, we hypothesize that the unpackaged part of the T. onnurineus NA1(T) genome might be related to the process that delivers DNA into ToEVs and/or the mechanism generating the ToEVs themselves.

Enhancing bio-hydrogen production from sodium formate by hyperthermophilic archaeon, Thermococcus onnurineus NA1.

Hyperthermophilic archaeon, Thermococcus onnurineus NA1 was reported to grow on formate producing hydrogen (H2). In this study, to sustain high H2 production rate and demonstrate the feasibility of mass production of H2, high cell density cultivation of T. onnurineus NA1 on sodium formate was employed under optimized conditions. From batch cultures, it was observed that the salinity of medium, significantly changed by the addition of formate salt and pH-adjusting agent, crucially affected cell growth and H2 production. With salinity carefully controlled between 3.7 and 4.6 %, 400 mM sodium formate was found to be an optimal initial concentration for maximizing cell growth-associated H2 production. Under optimal conditions, the repeated batch culture with cell recycling showed high cell density of OD600 of 1.7 in 3 and 30 L bioreactor, and the volumetric H2 production rate was enhanced up to 235.7 mmol L(-1) h(-1), which is one of the highest values reported to date.

A novel CO-responsive transcriptional regulator and enhanced H2 production by an engineered Thermococcus onnurineus NA1 strain.

Genome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism in Thermococcus onnurineus NA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes of Thermococcus species and "Candidatus Korarchaeum cryptofilum" OPF8. In-frame deletion of either corQ or corR caused a severe impairment in CO-dependent growth and H2 production. When corQ and corR deletion mutants were complemented by introducing the corQR genes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integrated corQR (ΔCorR/corQR(↑)) compared with those in the wild-type strain. In addition, the ΔCorR/corQR(↑) strain exhibited a much higher H2 production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2 production rate (191.9 mmol liter(-1) h(-1)) and the specific H2 production rate (249.6 mmol g(-1) h(-1)) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that the corQR genes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2 production.

Characterization of the frhAGB-encoding hydrogenase from a non-methanogenic hyperthermophilic archaeon.

The F420-reducing hydrogenase has been known as a key enzyme in methanogenesis. Its homologs have been identified in non-methanogenic hyperthermophilic archaea, including Thermococcus onnurineus NA1, but neither physiological function nor biochemical properties have been reported to date. The enzyme of T. onnurineus NA1 was distinguished from those of other methanogens and the members of the family Desulfurobacteriaceae with respect to the phylogenetic distribution of the α and ß subunits, organization of frhAGB genes and conservation of F420-coordinating residues. RT-qPCR and Western blot analyses revealed frhA gene is not silent but is expressed in T. onnurineus NA1 grown in the presence of sulfur, carbon monoxide, or formate. The trimeric enzyme complex was purified to homogeneity via affinity chromatography from T. onnurineus NA1 and exhibited catalytic activity toward the electron acceptors such as viologens and flavins but not the deazaflavin coenzyme F420. This is the first biochemical study on the function of the frhAGB-encoding enzyme from a non-methanogenic archaea.