• Journal of Microbiology and Biotechnology
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• 제26권1호
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• pp.89-98
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• 2016

Endophytes play an important role in the growth and development of the host. However, the study of endophytes is mostly focused on plants, and reports on bacteria associated with fungi are relatively rare. We studied the bacteria associated with fruiting bodies of Tricholoma matsutake picked from seven main T. matsutake-producing areas in Sichuan, China, by barcoded pyrosequencing. About 8,272 reads were obtained per sample, representing 40 phyla, 103 classes, and 495 genera of bacteria and archaea, and 361-797 operational taxonomic units were observed at a 97% similarity level. The bacterial community was always both more abundant and more diverse than the archaeal community. UniFrac analysis showed there were some difference of bacterial communities among the samples sites. Three bacterial phyla, Proteobacteria, Bacteroidetes, and Firmicutes, were dominant in all samples. Correlation analysis showed there was a significant correlation between some soil properties and bacterial community associated with T. matsutake. This study demonstrated that the bacteria associated with T. matsutake fruiting bodies were diversified. Among these bacteria, we may find some strains that can promote the growth of T. matsutake.

• Asian-Australasian Journal of Animal Sciences
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• 제12권1호
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• pp.129-138
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• 1999

If rumen bacteria can be manipulated to utilize nutrients (i.e., ammonia and plant cell wall carbohydrates) more completely and efficiently, the need for protein supplementation can be reduced or eliminated and the digestion of fiber in forage or agricultural residue-based diets could be enhanced. However, these approaches require a complete and accurate description of the rumen community, as well as methods for the rapid and accurate detection of microbial density, diversity, phylogeny, and gene expression. Molecular ecology techniques based on small subunit (SSU) rRNA sequences, nucleic acid probes and the polymerase chain reaction (PCR) can potentially provide a complete description of the microbial ecology of the rumen of ruminant animals. The development of these molecular tools will result in greater insights into community structure and activity of gut microbial ecosystems in relation to functional interactions between different bacteria, spatial and temporal relationships between different microorganisms and between microorganisms and reed panicles. Molecular approaches based on SSU rRNA serve to evaluate the presence of specific sequences in the community and provide a link between knowledge obtained from pure cultures and the microbial populations they represent in the rumen. The successful development and application of these methods promises to provide opportunities to link distribution and identity of gastrointestinal microbes in their natural environment with their genetic potential and in situ activities. The use of approaches for assessing pupulation dynamics as well as for assessing community functionality will result in an increased understanding and a complete description of the gastrointestinal communities of production animals fed under different dietary regimes, and lead to new strategies for improving animal growth.

• Journal of Microbiology and Biotechnology
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• 제25권5호
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• pp.569-578
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• 2015

Cold water paddy field soils are relatively unproductive, but can be ameliorated by supplementing with inorganic fertilizer from animal waste-based composts. The yield of two rice cultivars was significantly raised by providing either chicken manure or cow dung-based compost. The application of these composts raised the soil pH as well as both the total nitrogen and ammonium nitrogen content, which improved the soil's fertility and raised its nitrification potential. The composts had a measurable effect on the abundance of nitrogencycling-related soil microbes, as measured by estimating the copy number of various bacterial and archaeal genes using quantitative real-time PCR. The abundance of ammonia oxidizing archaea and bacteria was markedly encouraged by the application of chicken manure-based compost. Supplementation with the composts helped promote the availability of soil nitrogen in the cold water paddy field, thereby improving the soil's productivity and increasing the yield of the rice crop.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권4호
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• pp.344-350
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• 2006

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at $59^{\circ}C$ and pH 2. Along with another thermoacidophilic archaeon, Sulfolobus solfataricus, it is known to metabolize glucose by the non-phosphorylated Entner-Doudoroff (nED) pathway. In the course of these studies, the specific activities of glyceraldehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher in T. acidophilum than in S. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity from T. acidophilum cell extracts. The N-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49kDa and followed Michaelis-Menten kinetics with $K_m$ values of 0.56 and 0.32mM for DL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at $70^{\circ}C$. Of the seven sugars and four phosphate donors tested, only DL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as $Mn^{2+},\;CO^{2+},\;Ni^{2+},\;Zn^{2+},\;Ca^{2+},\;and\;Sr^{2+}$, were substituted for $Mg^{2+}$ the enzyme activities were less than 10% of that obtained in the presence of $Mg^{2+}$. The amino acid sequence of T. acidophilum glycerate kinase showed no similarity with E. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.

• BMB Reports
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• 제39권1호
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• pp.46-54
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• 2006

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of $11,180{\pm}50$ Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa $\alpha$ and 34 kDa $\beta$ subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at $55^{\circ}C$, pH 7.0. Maximum activity was observed at $70^{\circ}C$ and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with $K_m$ and $k_{cat}$ values of $163\;{\mu}M$ and $452\;min^{-1}$, respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.

• 한국지하수토양환경학회지:지하수토양환경
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• 제20권5호
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• pp.47-51
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• 2015

Biocatalytic degradation of total petroleum hydrocarbons (TPHs) in contaminated soil by hemoglobin and hydrogen peroxide is an effective soil remediation method. This study used a laboratory soil reactor experiment to evaluate the effectiveness of a nonspecific biocatalytic reaction with hemoglobin and H₂O₂ for treating TPH-contaminated soil. We also quantified changes in the soil microbial community using real-time PCR analysis during the experimental treatment. The results show that the measured rate constant for the reaction with added hemoglobin was 0.051/day, about 3.5 times higher than the constant for the reaction with only H₂O₂ (0.014/day). After four weeks of treatment, 76% of the initial soil TPH concentration was removed with hemoglobin and hydrogen peroxide treatment. The removal of initial soil TPH concentration was 26% when only hydrogen peroxide was used. The soil microbial community, based on 16S rRNA gene copy number, was higher (7.1 × 10⁶ copy number/g of bacteria, and 7.4 × 10⁵ copy number/g of Archaea, respectively) in the hemoglobin catalyzed treatment. Our results show that TPH treatment in contaminated soil using hemoglobin catalyzed oxidation led to the enhanced removal effectiveness and was non-toxic to the native soil microbial community in the initial soil.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1359-1367
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• 2005

The gene encoding Pyrobaculum arsenaticum DNA polymerase (Par DNA polymerase) was cloned and sequenced. The gene consists of 2,361 bp coding for a protein with 786 amino acid residues. The deduced amino acid sequence of Par DNA polymerase showed a high similarity to archaeal family B-type DNA polymerases (Group I), and contained all of the motifs conserved in the family B-type DNA polymerases for $3'{\rightarrow}5'$ exonuclease and polymerase activities. The Par DNA polymerase gene was expressed under the control of the T7lac promoter on the expression vector pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RP. The expressed enzyme was purified by heat treatment, and Cibacron blue 3GA and $Hirap^{TM}$ Heparin HP column chromatographies. The optimum pH of the purified enzyme was 7.5. The enzyme activity was activated by divalent cations, and was inhibited by EDTA and monovalent cations. The half-life of the enzyme at $95^{\circ}C$ was 6 h. Par DNA polymerase possessed associated $3'{\rightarrow}5'$ proofreading exonuclease activity, which is consistent with its deduced amino acid sequence. PCR experiment with Par DNA polymerase showed an amplified product, indicating that this enzyme might be useful in DNA amplification and PCR-based applications.

• Journal of Microbiology and Biotechnology
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• 제26권5호
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• pp.867-875
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• 2016

Archaea substantially contribute to global geochemical cycling and energy cycling and are impacted by land-use change. However, the response of archaeal communities to a change from upland field to paddy field has been poorly characterized. Here, soil samples were collected at two depths (0-20 cm and 20-40 cm) from one upland field and six paddy fields that were established on former upland fields at different times (1, 5, 10, 20, 30, and 40 years before the study). Barcoded pyrosequencing was employed to assess the archaeal communities from the samples at taxonomic resolutions from phylum to genus levels. The total archaeal operational taxonomic unit (OTU) richness showed a significant positive correlation with the land-use change duration. Two phyla, Euryarchaeota and Crenarchaeota, were recorded throughout the study. Both the relative abundance and OTU richness of Euryarchaeota increased at both depths but increased more steadily at the subsurface rather than at the surface. However, these data of Crenarchaeota were the opposite. Additionally, the archaeal composition exhibited a significant relationship with C/N ratios, total phosphorus, soil pH, Olsen phosphorus, and the land-use change duration at several taxonomic resolutions. Our results emphasize that after a change from upland fields to paddy fields, the archaeal diversity and composition changed, and the duration is an important factor in addition to the soil chemical properties.

• 한국콘텐츠학회논문지
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• 제20권8호
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• pp.674-684
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• 2020

하수의 종류(생활하수, 축산폐수) 및 다양한 처리 공정에 따른 미생물군집구조를 비교하기 위해 A2O공법으로 운영되는 생활하수처리시설(안심·서부·신천)의 10개 생물학적 반응조와 축산폐수처리시설의 활성슬러지를 채취해 DNA 유전체를 추출한 후 세균은 프라이머 27F/518R, 고세균의 경우, 프라이머 Arch519F/A958R를 이용해 유전체를 증폭시켰고 그 염기서열을 Roche 454 GS-FLX Titanium을 이용한 pyrosequencing 법으로 분석하였다. 그 결과, 생활하수와 축산폐수에 따른 미생물 군집구조의 차이는 컸지만 A2O공법에 따른 산소 유무 등의 환경 변화와 관련된 군집구조의 변화는 크지 않았다. 혐기조 및 무산소조 반응조들에서만 분석한 고세균 군집 결과에서는 동일 하수처리시설의 반응조들의 고세군군집들끼리만 모이는 하수처리시설별 집괴현상을 나타냈다. 세균다양성 및 종 풍부도가 높은 서부처리시설이 다른 시설보다 더 높은 질소 및 인 제거율을 나타냈다.

• Journal of Microbiology and Biotechnology
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• 제16권11호
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• pp.1826-1831
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• 2006

A novel hyperthermophilic, anaerobic, heterotrophic archaeon, designated strain $NA1^T$, was isolated from a deep-sea hydrothermal vent area (depth, 1,650 m) within the Papua New Guinea-Australia-Canada-Manus (PACMANUS) field. Cells of this strain were motile by means of polar flagella, coccoid-shaped with a diameter of approximately $0.5-1.0{\mu}m$, and occurred as single cells. Optimal temperature, pH, and NaCl concentration for growth were $80^{\circ}C$, 8.5, and 3.5%, respectively. The new isolate was an obligate heterotroph that utilized yeast extract, beef extract, tryptone, peptone, casein, and starch as carbon and energy sources. Elemental sulfur was required for growth and was reduced to hydrogen sulfide. The G+C content of the genomic DNA was 52.0 mol%. Phylogenetic analysis of the 16S rRNA gene indicated that strain $NA1^T$ belongs to the genus Thermococcus, and the organism is most closely related to T. gorgonarius, T. peptonophilus, and T. celer; however, no significant homology was observed among species by DNA-DNA hybridization. Strain $NA1^T$ therefore represents a novel species for which the name Thermococcus onnurineus sp. novo is proposed. The type strain is $NA1^T$ (=KCTC 10859, =JCM 13517).