· 2016. 5. 24. · Poster | 1 A001 Paenibacillus baekrokdamisoli sp., nov., Isolated from Soil of Crater

Poster

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A001

Paenibacillus baekrokdamisoli sp., nov., Isolated from Soil of Crater Lake

Keun Chul Lee1, Kwang Kyu Kim1, Jong-Shik Kim2, Dae-Shin Kim3, Suk-Hyung Ko3, Seung-Hoon Yang3, and Jung-Sook Lee1,4*1KCTC/KRIBB, 2GIMB, 3World Heritage and Mt. Hallasan Research Institute, 4UST

A novel bacterial strain Back-11T was isolated from sediment soil of crater lake, Baekrokdam, Hallasan, Jeju, Republic of Korea. Cells of strain Back-11T were Gram-staining-positive, motile, endospore-forming, rod-shaped and oxidase- and catalase-positive. It contained anteiso-C15:0 as the major fatty acids, menaquinone-7 (MK-7) as the predominant isoprenoid quinone, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and four unidentified aminophospholipids as the main polar lipids, and meso-DAP as the diagnostic diamino acid in the cell-wall peptidoglycan. The DNA G+C content was 45.3 mol%. Phylogenetic analysis, based on 16S rRNA gene sequencing, showed that strain Back-11T was most closely related to Paenibacillus taihuensis THMBG22T (95.5%) and fell into a clade in the genus Paenibacillus. On the basis of phylogenetic, chemotaxonomic and phenotypic data, strain Back-11T represents a novel species in the genus Paenibacillus, for which the name Paenibacillus baekrokdamisoli sp. nov. is proposed, with strain Back-11T (= KCTC 33723T = CECT 8890T) as the type strain.

A002

Rhodanobacter aciditrophus sp. nov., an Acidophilic Bacterium Isolated from Mine Wastewater

Hyeon-Woo Koh, Sudas Rani, and Soo-Je Park*

Department of Biology, Jeju National University

A novel strain (designated sjH1T), characterized as aerobic, Gram-stain negative, oxidase-positive, catalase-negative, motile and rod-shaped, was isolated from mine wastewater. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain sjH1T belonged to the genus Rhodanobacter. Strain sjH1T was closely related to Rhodanobacter thiooxydans LCS2T (98.0% 16S rRNA gene sequence similarity), Rhodanobacter denitrificans 2APBS1T

(97.7%), Rhodanobacter soli DCY45T (97.2%), and Rhodanobacter caeni MJ01T (97.0%). The DNA G+C content of strain sjH1T was 69.2 mol%. DNA-DNA relatedness (<60%) indicated that strain sjH1T represents a distinct species that is separate from R. thiooxydans, R. denitrificans, R. soli, and R. caeni. The major ubiquinone was Q-8, and major fatty acids were summed feature 9 (iso–C17:1 ω9c and/or C16:0 10–methyl), iso–C15:0, iso–C17:0, iso–C16:0, and anteiso–C15:0. Based on data from this polyphasic study, it is proposed that sjH1T (=KCTC 42660T =JCM 30774T) is the type strain of a novel species, Rhodanobacter aciditrophus sp. nov.[Supported by grants from NRF (2015R1C1A1A01053750 & 2015M3D 3A1A01064881)]

A003

Lentibacillus kimchii sp. nov., an Extremely Halophilic Bacterium Isolated from Kimchi

Young Joon Oh1, Hae-Won Lee2, Seul Ki Lim1, Min-Sung Kwon1, Jieun Lee1, Ja-Young Jang1, Jong Hee Lee1, Hae Woong Park3, Seong Woon Roh4, and Hak-Jong Choi1*1Microbiology and Functionality Research Group, World Institute of Kimchi 2Hygienic Safety and Analysis Center, World Institute of Kimchi 3Advanced Process Technology Research Group, World Institute of Kimchi4Biological Disaster Analysis Group, Korea Basic Science Institute

A Gram-stain positive, aerobic, non-motile and extremely halophilic bacterium, designated strain K9T, was isolated from kimchi. The strain was observed to be endospore-forming rod-shaped cells showing oxidase- and catalase- positive reactions. Strain K9T was able to grow at 10.0–30.0% (w/v) NaCl (optimum, 15.0–20.0%), pH 7.0–8.0 (optimum, pH 7.5) and 15–40°C (optimum, 30°C). The polar lipids of strain K9T consisted of phosphatidylglycerol, two unidentified phospholipids and an unidentified glycolipid. The predominant isoprenoid quinone was menaquinone-7. The major cellular fatty acids (>20% of the total) were anteisio-C15:0 and anteisio-C17:0. The cell wall peptidoglycan of strain K9T was determined as meso-diaminopimelic acid. The G+C content of genomic DNA was 48.2 mol%. Phylogenetic analysis based on the 16S rRNA genes revealed that the isolated strain was most closely related to the Lentibacillus salinarum AHS-1T with a similarity of 96.63%. Based on its phenotypic, chemotaxonomic and phylogenetic data, strain K9T is considered to represent a novel species belonging to the genus Lentibacillus, for which the name Lentibacillus kimchii sp. nov. is proposed. The type strain is K9T (=KACC 15543T =JCM 17730T).[This research was supported by a grant from the World Institute of Kimchi funded by the Ministry of Science, ICT and Future Planning (KE1601-2), Republic of Korea.]

A004

Study on the Mycelium and Morphological Characteristic of Naematoloma sublateritium

Jongwoon Choi, Hasun Kim, Dongyong Shin, and Yunkyeong Lee*

Forest Research Institute of Gangwon-do

This study showed to excellent mycelium growing at Glucose in circular the carbon which was suitable for the third, mycelium growing of a N. sublateritium provisional results carbon circle regarding a nitrogen circle, and cultures of a N. sublateritium looked at malt extract in case of organic nitrogen circles. However, a kind circular carbon nitrogen used to a test is restrictive, and provisional shall consist of various elements by a foundation. This study looked in a phosphoric acid circle to affect the fourth, a mycelium of a N.sublateritium to growth and development. Also, If this study added it was investigated p-aminobenzoic acid in a vitamin circle so that hypha growing was comparatively good.

2016 International Meeting of the Microbiological Society of Korea

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A005

A Multiplex-PCR for Rapid Identification of 4 Enterococcus Species Using Species-Specific Primers from Comparative Genomics

Jongbin Park1, Gwi-Deuk Jin2, Yong Hyun Kim2, Jae In Pak2,3, and Eun Bae Kim2,3*1Department of Animal Life System, College of Animal Life Sciences, Kangwon National University, 2Department of Animal Life Science, College of Animal Life Sciences, Kangwon National University, 3Division of Applied Animal Science, College of Animal Life Sciences, Kangwon National University

Enterococcus species are lactic acid bacteria frequently found in food/feed and human/animal guts. They are regarded as either commensal bacteria or sometimes pathogens. For these reasons, rapid identification is required in both food industries and clinical practices. Here, we investigated a new rapid identification method based on species-specific genes from comparative genomics for 4 Enterococcus species, 16S rRNA sequences of which are similar. From genome comparison, we identified species-specific genes for Enterococcus faecium, Enterococcus faecalis, Enterococcus durans and Enterococcus hirae, respectively. Four sets of primers were designed for each species. After each primer set was tested and optimized for identification of corresponding species, the four primers were re-optimized for Multiplex- PCR using already known Enterococcus strains. This method was re-confirmed using locally isolated strains of 4 different species from chicken feces, cow feces, horse feces, meats, and fermented soybean. In this study, we designed a species-specific primers distinguishing the 4 Enterococcus spp. in our new Multiplex-PCR method. This will help rapid decision making to get practical benefits in food/feed industries and clinical services.[This study was supported by the Strategic Initiative for Microbiomes in Agriculture and Food, Republic of Korea (914005-04).]

Keywords: Enterococcus, Species-specific primer, Multiplex-PCR, Rapid identification, 16S rRNA

A006

Investigating the Inflammatory and Phenotypic Responses of Multiple Cultured Human Epithelial Cells to Predatory Bacteria

Wasimul Bari and Ajay K. Monnappa*

Ulsan National Institute of Science and Technology

Bdellovibrio-and-like-organisms (BALOs) are small Gram-negative predatory bacteria that attack and kill other Gram-negative bacteria including many pathogens. Although BALOs are considered to be a beguiling alternative to antibiotics for treating multi-drug resistant infections, the possible inflammatory and stress response in human cells to predatory bacteria remains to be explored. In this study, human intestinal (T84), alveolar (NuLi-1) and breast (MCF10a) epithelial cells and the mouse Raw 264.7 macrophage cell line were exposed to predatory bacteria, Bdellovibrio bacteriovorus HD100, B. bacteriovorus BY1 and Bacteriovorax stolpii EB1, to determine if they elicit inflammatory responses. However, none of the human epithelial cells produced pro-inflammatory IL-8 and TNF-α or anti-inflammatory IL-6 and IL-10 cytokines in response to the predatory bacteria, whereas TNF-α was produced by the mouse macrophage cell line. In contrast, exposure to a Gram-negative E. coli strain led to significantly higher amount of IL-8 and TNF-α from three human cell lines and a mouse cell line, respectively. While, none of the predator strains were cytotoxic, lack of response was supported by confocal microscopy results that showed the predatory bacterial strains did not bring about any F-actin rearrangement in the mammalian cells. This in vitro study illustrates that predatory bacteria are not harmful to human epithelial cells and adds valuable insight for future in vivo studies.

A007

Flavihumibacter sediminis sp. nov., Isolated from Tidal Flat Sediment

Do-Hoon Lee and Chang-Jun Cha*

Department of Systems Biotechnology, Chung-Ang University

A yellow-colored, Gram-stain-positive, aerobic, rod-shaped and non-motile bacterial strain, designated AM3T, was isolated from the tidal flat in Ganghwa-do, South Korea. Strain AM3T optimally grew on R2A at 30oC and pH 7.0 and did not require NaCl for growth. Phylogenetic analysis based on 16S rRNA gene sequence similarity showed that strain AM3T belonged to the genus Flavihumibacter within the family Chitinophagaceae and was the most closely related to Flavihumibacter cheonanensis KACC 17467T (98.3%), followed by Flavihumibacter solisilvae KACC 17917T (97.4%). DNA-DNA relatedness levels of strain AM3T were 30.6% to F. cheonanensis KACC 17467T and 42.6% to F. solisilvae KACC 17917T. The major isoprenoid quinone was menaquinone (MK-7). The predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The G+C content of the genomic DNA was 47.7 mol%. On the basic polyphasic taxonomic data, strain AM3T represents a novel species in the genus Flavihumibacter, for which name Flavihumibacter sediminis sp. nov. is proposed.

A008

Taxonomic Study of the Genus Hymenobacter

Joo Won Kang, Mi Sun Kim, Ji Hee Lee, Seon Choi, Da Hyun Kim, and Chi Nam Seong*

Department of Biology, College of Life Science and Natural Resources, Sunchon National University

The genus Hymenobacter, a member of the family Cytophagaceae (Stanier, 1940), phylum Bacteroidetes, was proposed to group members producing large amounts of extracellular polymeric substances and spreading in thin, pinkish layers on agar surfaces (Hirsch et al., 1999; Buczolits et al., 2006). At the time of writing, the genus comprises 36 species with Hymenobacter roseosalivarius as a type species. The members of the genus Hymenobacter species have been isolated from a wide range of sources including air, soil, fresh water, estuarine water, extreme conditions such as arid land, glacier, uranium mine waste water treatment, pork, tidal flat and basal ice. Common characteristics of Hymenobacter species are Gram-stain-negative, non-spore-forming, rods and producing large amounts of extracellular polymeric substances (EPS). Major fatty acids were iso-C15:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B). The major respiratory quinone is menaquinone 7 (MK-7) and polyamine is sym-homospermidine. The G+C content of the DNA varies between 53 and 70 mol%.[This research was supported by the project on survey and excavation of Korean indigenous species of the National Institute of Biological Resources (NIBR) under the Ministry of Environment, Republic of Korea. It was also supported by the CK(university for Creative Korea)-Ⅰ]

Poster

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A009

OrthoANI: An Improved Algorithm and Software for Calculating Average Nucleotide Identity

Imchang Lee1,2, Yeong Ouk Kim2,3, Sang-Cheol Park2,3, and Jongsik Chun1,2,3*1School of Biological Sciences, Seoul National University2Institute of Molecular Biology & Genetics, Seoul National University3Interdisciplinary Program in Bioinformatics, Seoul National University

Species demarcation in Bacteria and Archaea is mainly based on overall genome relatedness, which serves a framework for modern microbiology. Current practice of obtaining these measures between two strains is shifting from experimentally determined similarity obtained by DNA-DNA hybridization (DDH) to genome sequence-based similarity. Average nucleotide identity (ANI) is a simple algorithm that mimics DDH. Like DDH, ANI values between two genome sequences may be different from each other when reciprocal calculations are compared. We compared 63,690 pairs of genome sequences and found that the differences in reciprocal ANI values are significantly high, showing over 1% in some cases. To resolve this problem of not being symmetrical, new algorithm, named OrthoANI, was developed to accommodate the concept of orthology for which both genome sequences were fragmented and only orthologous fragment pairs taken into consideration for calculating nucleotide identities. OrthoANI is highly correlated with ANI (using BLASTn) and the former showed ~0.1% higher values than the latter. In conclusion, OrthoANI provides a more robust and faster means of calculating average nucleotide identity for the taxonomic purposes. The standalone software tools are freely available at http://www.ezbiocloud. net/sw/oat.[Supported by National Science Foundation]

A010

A Bacterium Representing Novel Species in the Genus Sphingomonas, Isolated from Freshwater of Juam Reservoir

Ji Hee Lee, Dae In Kim, Yong Seob Joo, Seo Young Kim, and Chi Nam Seong*


A motile, aerobic and light yellow pigmented bacterium, designated strain 03SUJ6T was isolated from the freshwater of Juam reservoir, Republic of Korea. Cells were Gram-stain-negative, catalase and oxidase activities are negative. Strain 03SUJ6T grow at 15-37°C (optimally at 25-30°C) and at pH 6.0-8.0 (optimally at pH 7.0). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 03SUJ6T formed a distinct lineage within the genus Sphingomonas and was most closely related to Sphingomonas daechungensis KCTC 23718T (97.44% 16S rRNA gene sequence similarity), S. ginsengisoli KCTC 12630T (97.29%), S. humi KCTC 12341T (97.15%) and S. rosea KCTC 12339T (97.01%). The DNA-DNA relatedness ratios were lower than 70%. The predominant fatty acids are C16:1, C17:1 ω6c, Summed feature 3 (C16:1 ω7c/C16:1 ω6c and/or C16:1 ω6c/C16:1 ω7c) and Summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The major respiratory quinone was ubiquinone 10 (UK-10) and the major polyamine is sym-homo-spermidine. The DNA G+C content of the genomic DNA was 64.7 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain 03SUJ6T can be considered as a novel species of the genus Sphingomonas.[This research was supported by the project on survey and excavation of Korean indigenous species of the National Institute of Biological Resources (NIBR) under the Ministry of Environment, Republic of Korea. It was also supported by the CK(university for Creative Korea)-I]

A011

Aquimarina 820-2 sp. nov., Isolated from Marine Sponge Dysidea sp.

Ga Eun Lee and Jin Sook Park*

Department of Biological Science and Biotechnology, Hannam University

An orange, rod-shaped bacterium, designated strain Aquimarina 820-2, was isolated from the marine sponge Dysidea sp., collected from Micronesia. Cells were Gram-stain -negative, and catalase- and oxidase-positive. Optimal growth was observed at 30°C, at pH 9, and with NaCl 3% (w/v). The major fatty acids were iso-C15:0, iso-C17:0 3-OH, iso-C15:0 3-OH and iso-C15:0. Isoprenoid quinone was menaquinone. The DNA G+C content was 30.6 mol%. 16S rRNA gene sequence analysis showed that strain Aquimarina 820-2 belonged to genus Aquimarina, the closest member being Aquimarina mytili PSC33T, with a gene sequence similarity of 96.8%. A number of phenotypic characteristics distinguished strain Aquimarina 820-2 from recognized members of the genus Aquimarina. On the basis of data presented in this study, strain Aquimarina 820-2 is considered to represent a novel species of the genus Aquimarina.[Supported by NRF and Ministry of Oceans and Fisheries]

A012

Acetobacter oryzifermentans sp. nov., Isolated from a Korea Traditional Vinegar

Ga Youn Cho and Che Ok Jeon*

Department of Life Science, Chung-Ang University

An acetic acid bacterium, designated SLV-7T, showing great ethanol and acetate resistance was isolated from a Korean traditional vinegar. The cells were Gram-staining-negative, catalase-positive, oxidase-negative, and non-motile rods. The G+C content was 52.4 mol%. Strain SLV-7T was most closely related to Acetobacter pasteurianus subsp. pasteurianus LMG 1262T, A. pasteurianus subsp. ascendens LMG 1590T, A. pasteurianus subsp. paradoxus LMG 1591T, and A. pomorum LHT 2458T with the 16S rRNA gene sequence similarities of 99.9%, 99.8%, 99.7%, and 99.7%, respectively, but the DNA–DNA relatedness values between strain SLV-7T and the close related strains were 43.4 ± 8.6%, 21.8 ± 3.6%, 27.9 ± 5.7% and 25.2 ± 4.5%, respectively. Phylogenetic analysis using the concatenated sequences of dnaK, groEL and rpoB indicated that the isolate formed a cluster with A. pasteurianus subsp. ascendens, but was distinct from other Acetobacter species. Average nucleotide identity (ANI) values of strain SLV-7T with other genome sequenced Acetobacter species including closely related type strains were less than 90.52%. On the basis of genotypic, chemotaxonomic and molecular features, strain SLV-7T clearly represents a novel species of the genus Acetobacter, for which the name Acetobacter oryzifermentans sp. nov. is proposed. The type strain is SLV-7T. [This work was spported by the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ00999302)”, RDA]


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A013

Lapsobacter soli gen. nov., sp. nov., Isolated from Soil of a White Heron Nesting Site

Min-Kyeong Kim1, Yujin Choi1, Tae-Su Kim1,2, Ji-Hye Han1,3, Yochan Joung1,4, and Seung Bum Kim1*1Department of Microbiology and Molecular Biology, College of Bioscience and Biotechnology, Chungnam National University, 2Clinical Drug Manufacturing Center, Osong Medical Innovation Foundation, 3Bacterial Resources Research Team, Freshwater Bioresources Research Division, Nakdonggang National Institute of Biological Resources, 4Department of Biology, Inha University

A Gram-stain-negative, motile by gliding, non-spore-forming and short rod-shaped bacterial strain, designated R1-15T, was isolated from soil of a white heron nesting site (36° 21′ 52″ N, 127° 20′ 59″ E) and its taxonomic position was evaluated using a polyphasic approach. Strain R1-15T grew at 15–37°C (optimum 30°C), at pH 6–7 (optimum pH 6) and in the presence of 0–1% (w/v) NaCl (optimum 0%) on 0.1× TSA. On the basis of 16S rRNA gene sequence similarity, the novel strain was assigned to the family Chitinophagaceae of the class Bacteriodetes, and its closest related taxa were species of the genera Taibaiella (89.98–88.76% sequence similarity), Chitinophaga (89.80– 87.38%), Lacibacter (89.78–89.24%), Parasegetibacter (89.38–88.06%) and Flavitalea (89.17–88.66%). Flexirubin-type pigments were produced. The only isoprenoid quinone was MK-7, and the major polar lipid was phosphatidylethanolamine. The genomic DNA G+C content of the strain was 43.8 mol%. The phylogenetic, chemotaxonomic and phenotypic data supported the affiliation of strain R1-15T to a novel species of new genus within the family Chitinophagaceae, for which the name Lapsobacter soli gen. nov., sp. nov. is proposed. The type strain is R1-15T (= JCM 31190T).

A014

Flavobacterium keumense sp. nov., Isolated from Freshwater

Adaeze Ekwe, Joong-hyeon Ahn, and Seung Bum Kim*

Department of Microbiology and Molecular Biology, Chungnam National University

A yellow-pigmented, oxidase positive, catalase negative, Gram-negative, non-spore-forming, rod-shaped, aerobic, non-motile bacterial strain designated K3R-10T was isolated from a freshwater source in Korea. The strain grew over a temperature range of 4oC to 35oC (optimum = 30oC) and pH range 6-7 (optimum = 7). No growth was observed above 0.5% NaCl. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain K3R-10T belongs to the genus Flavobacterium and shares close similarities with Flavobacterium succinicans (97.02%), Flavobacterium chungangense (96.39%), Flavobacterium branchiophilum (96.39%) and Flavobacterium piscis (96.32%). The polar lipid profile revealed the presence of phosphatidylethanolamine, an unknown aminolipid and 3 unknown phospholipids. The DNA G+C content was 35.4mol%, MK-6 was the major quinone and homospermidine was the predominant polyamine. Absence of an aminophospholipid in its polar lipid profile, ability to hydrolyse gelatin, acid production from carbohydrates, G+C content and colony morphology are some of the properties that differentiate Flavobacterium strain K3R-10T from related species. Thus, on the basis of phenotypic, chemotaxonomic and phylogenetic differences, strain K3R-10T represents a novel species in the genus Flavobacterium, for which the name Flavobacterium keumense sp. nov. is proposed. The type strain is K3R-10T (NCBI Accession number = KC355348).

A015

Thalassotalea litorea sp. nov., Isolated from Seashore Sand

Heeyoung Kang, Haneul Kim, and Kiseong Joh*

Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies

An aerobic, Gram-strain-negative, rod-shaped bacterium designated HMF4135T was isolated from a sand sample collected from the seashore of the East Sea, Korea. Comparative 16S rRNA gene sequence analysis showed that strain HMF4135T belonged to the genus Thalassotalea and was most closely related to Thalassotalea ponticola (96.4% sequence similarity). Strain HMF4135T was characterized by the predominance of C16:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:1 ω9c and C12:0 3-OH. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. DNA G+C content was 41.9 mol%. Based on the distinctive phenotypic characteristics and phylogenetic analysis, it is concluded that strain HMF4135T represents a novel species of the genus Thalassotalea, for which the name Thalassotalea litorea sp. nov. is proposed. The type strain of the species is strain HMF4135T (= KCTC 52154T = CECT -ingT).

A016

Roseovarius salarius sp. nov., Isolated from a Solar Saltern

Haneul Kim1, Heeyoung Kang1, Yochan Joung2, and Kiseong Joh1*1Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, 2Department of Biological Sciences, Inha University

A Gram-staining negative and rod-shaped bacterial strain, designated HMF2641T, was isolated from a solar saltern in Korea. A phylogenetic tree based on 16S rRNA gene sequences showed that strain HMF2641T formed a lineage within the genus Roseovarius. Strain HMF2641T was closely related to Roseovarius tolerans EL-172T (96.1% sequence similarity), R. azorensis SSW084T (95.7%) and R. mucosus DSM 17069T (94.7%). The major fatty acids of strain HMF2641T were summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c), cyclo-C19:0 ω8c and C16:0. DNA G+C content of strain HMF2641T was 65.9 mol%. On the basis of the evidence presented in this study, strain HMF2641T represents a novel species of the genus Roseovarius, for which the name Roseovarius salarius sp. nov., is proposed the type strain HMF2641T (=KACC 18421T =CECT 8857T).

Poster

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A017

Zeaxanthinibacter aestuarii sp. nov., Isolated from Estuary Sediment and Emended Description of the Genus Asker2007

Yun Hee Lee , Hye Im Jeong, Sang Eun Jeong and Che Ok Jeon*


A novel Gram-staining-negative, strictly aerobic, non-motile bacterium and yellow pigmented bacterium, designated strain S2-22T, was isolated from an estuary sediment in South Korea. Cells of strain S2-22T were oxidase- and catalase-positive rods without gliding motility. Growth was observed at 15–43°C (optimum, 37°C), at pH 5.5–9.0 (optimum, pH 6.5–7.5) and in the presence of 0.0–10.0% (w/v) NaCl (optimum, 2.0%). The respiratory quinone was only detected Menaquinone 6 (MK-6) and iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH and summed feature 3 (comprising C16:1 ω7c/C16:1 ω6c and/or C16:1 ω6c/C16:1 ω7c) were found out as the major fatty acids. The major polar lipid was phosphatidylethanolamine and five unidentified aminolipids, an unidentified phospholipid, and three unidentified lipids were also detected as the minor polar lipids. The G+C content of the genomic DNA was 45.5 mol %. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain S2-22T formed a tight phyletic lineage with Zeaxanthinibacter enoshimensis TD-ZE3T with a high bootstrap value and their 16S rRNA gene sequence similarity was 94.6 %. Based on the phenotypic, chemotaxonomic and molecular features, strain S2-22T clearly represents a novel species of the genus Zeaxanthinibacter, for which the name Zeaxanthinibacter aestuarii sp. nov. is proposed. The type strain is S2-22T (=KACC 18503T =JCM 31155T).

A018

A First Report of Pseudoalteromonas tetraodonis Isolated from Apostichopus japonicas Guts

Hyunjun Choi, Jihoon Jo, and Chungoo Park*

School of Biological Sciences and Technology, Chonnam National University

More than 1,250 species of sea cucumber have been identified from the sea floor worldwide and approximately 20 of them are edible. Sea cucumbers represent commercially and medically valuable members of the group Echinodermata, which itself is one of the most abundant and ecologically successful marine invertebrate clades. One economically important species as a source of seafood and traditional medicine is the sea cucumber Apostichopus japonicus Selenka 1867, which is mainly found off the coasts of northeast Asia. Moreover, salted sea cucumber intestines (know as konowata in Japanese) have been considered as a gourmet and healthy food. To explore whether there are beneficial microorganisms in sea cucumber guts, we screened culturable microorganisms from guts of A. japonicus, and isolated Alteromonas sp., Pseudoalteromonas sp., Vibrio sp., and Shewanella sp.. Because it has been well known that Pseudoalteromonas tetraodonis has antibacterial, bacteriolytic, agarolytic and algicidal activities, we tested antibacterial activity using paper disc diffusion method about our newly isolated Pseudoalteromonas tetraodonis CSB01KR in A. japonicus guts. Carried out the antibacterial activity test against human pathogens, the newly isolated Pseudoalteromonas tetraodonis CSB01KR has potent antibacterial activity. As a result, the Pseudoalteromonas tetraodonis CSB01KR may have considerable research value in medical and pharmaceutical fields.

A019

A New Record of Didymella pinodella Isolated from a Fruit of Red Pepper in Korea

Tham Thi Duong and Hyang Burm Lee*

Division of Food Technology, Biotechnology & Agrochemistry, College of Agriculture & Life Sciences, Chonnam National University

A fungal strain EML-RPF-3 was isolated from a fruit of red pepper (Capsicum annuum L.) in Korea. Pycnidia was solitary, formed on the agar surface or submerged, variable in shape and size, subglobose or elongated. Surface of pycnidial wall was glabrous. A cirrhus on pycnidia was blackish and frequently observed on PDA medium. Pycnidiospores were hyaline, mostly non-septate, sometimes 1-septate, ellipsoid, few ovoid, measured 2.0-3.2 µm in diameter. Chlamydospores were dark brown, spherical to irregular, smooth to rough, terminal or intercalary and produced singly or in chains. BLASTn search of the rDNA ITS sequences via NCBI database indicated that the isolate matched Didymella pinodella (GenBank accession numbers, KM030324 and KM030323) with identity values of 99.9% (992/993 bp) and 99.9% (982/983 bp), respectively. Based on the morphological characteristics and sequence analysis of the internal transcribed (ITS) regions, the isolate was identified as Didymella pinodella belonging to Pleosporales. To our knowledge, the species of Didymella pinodella represents a new record in Korea.

A020

Pseudahrensia todarodis sp. nov., a Novel Bacterium Isolated from the Gut of a Japanese Flying Squid

Hyun Sik Kim, Pil Soo Kim, Dong-Wook Hyun, June-Young Lee, Woorim Kang, Na-Ri Shin, Tae Woong Whon, and Jin-Woo Bae*

Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University

A novel Gram-stain-negative, non-motile, non-spore-forming, non-flagellated, aerobic, beige-coloured and rod-shaped bacterium, designated strain KHS02T, was isolated from the gut of a Japanese flying squid, Todarodes pacificus, collected from the East Sea, Korea. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain KHS02T formed a monophyletic clade with P. aquimaris HDW-32T, with which it had the highest sequence similarity (98.67%). Strain KHS02T grew optimally at pH 7 with 2% (w/v) NaCl at 25°C on marine broth 2216, and could not grow without Na+. The predominant isoprenoid quinone was ubiquinone-10. The major fatty acids (more than 10% of total fatty acids) were summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c, 65.92%) and 11-methyl C18 : 1 ω7c (10.54%). The polar lipids of strain KHS02T comprised phosphatidylcholine, phosphatidylglycerol, phosphatidyle-thanolamine, two unidentified aminolipids, an unidentified phospholipid and an unidentified lipid. The genomic DNA G+C content was 58.6 mol%. DNA-DNA hybridisation showed that the isolate shared 16.2% ± 1.3% (reciprocal, 15.7% ± 2.8%) genomic relatedness with the type strain of the closest species. In conclusion, we suggest this isolate is a novel species of the genus Pseudahrensia, for which the name Pseudahrensia todarodis is proposed. The type strain is KHS02T (KACC 18257T = JCM 30419T). [This work was supported by grants from the NRFK (2011-0028854 and 2015H1A2A1034891) and NIBR (2013-02-001).


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A021

A Novel Microbulbifer-like Bacteria Isolated from the Gut of Purple Sea Urchin, Heliocidaris crassispina

June-Young Lee1,2, Pil Soo Kim1,2, Dong-Wook Hyun1,2, Hyun Sik Kim1,2, Na-Ri Shin1,2, Mi-Ja Jung1,2, Ji-Hyun Yun1,2, Min-Soo Kim1,2, Tae Woong Whon1,2, and Jin-Woo Bae1,2*1Department of Life and Nanopharmaceutical Sciences, 2Department of Biology, Kyung Hee University

A novel Gram-negative, strictly aerobic bacterium, designated as strain AM134T, was isolated from the gut of a purple sea urchin Heliocidaris crassispina which was collected from the coastal waters of Dokdo, Korea. The 16S rRNA gene sequence analysis showed that strain AM134T belonged to the genus Microbulbifer in the family Alteromonadaceae and identified that the isolate had the highest sequence similarity with Microbulbifer epialgicus F-104T (98.90% similarity). Strain AM134T grew optimally at 30˚C, in the presence of 2% (w/v) NaCl and at pH 7. The G+C content of genomic DNA was 56.1 mol%. The DNA-DNA hybridization analysis showed the strain shared less than 27% genomic relatedness with Microbulbifer epialgicus F-104T. The polar lipid profiles of strain AM134T were constituted of phosphatidylethanolamine, phosphatidylserine, three unidentified amino-phospholipids, two unidentified phospholipids, an unidentified amino lipid and six unidentified lipids. The major cellular fatty acids were summed feature 8 (C18:1 ω6c and/or C18:1 ω7c) and C16:0. The major respiratory quinone was identified as ubiquinone-8 (Q-8). The results of the phylogenetic, phenotypic and genotypic analyses suggest that strain AM134T represents a novel species in the genus Microbulbifer, for which the name Microbulbifer echini is proposed, where the type strain is AM134T (=KACC 18258T =JCM 30400T). [Supported by grants from the Mid-career Researcher Program (2011- 0028854).]

A022

Flexivirga lutea sp. nov., an Actinobacterium Isolated from the Stool of a Crested Ibis

Woorim Kang, Dong-Wook Hyun, Pil Soo Kim, Na-Ri Shin, Hyun Sik Kim, June-Young Lee, Euon Jung Tak, and Jin-Woo Bae*


A novel Gram-staining-positive, aerobic, non-motile and coccus-shaped bacterium, designated strain TBS-100T, was isolated from the intestine of a crested ibis, Nipponia nippon. The phylogenetic analysis based on the 16S rRNA gene sequences showed that the nearest species of TBS-100T was Flexivirga alba DSM 24460T with 97.11% similarity and strain TBS-100T belonged to the genus Flexivirga. The optimum growth condition for strain TBS-100T was 30°C, at a pH of 7 and 0% (w/v) NaCl. The primary cellular fatty acids of strain TBS-100T were anteiso-C17:0 and iso-C17:0. The predominant isoprenoid quinone was MK-8 (H4, 70.2%) and MK-8 (H6, 29.7%). The polar lipids were diphosphatidylglycerol, phosphatidylinositol, seven unidentified lipid and an unidentified phospholipid. The whole cell-wall sugars of strain TBS-100T were ribose, glucose, galactose, rhamnose and mannose. The peptidoglycan contained alanine, lysine, glutamine, glycine and aspartic acid. The DNA G+C content was 64.8 mol%. The phenotypic, phylogenetic, and genotypic analyses indicated that strain TBS-100T represents a novel species of the genus Flexivirga for which the name Flexivirga lutea sp. nov. is proposed.[This work was supported by grants from the Mid-career Researcher Program (2011-0028854) through the National Research Foundation of Korea, funded by the Ministry of Education of Korea and the National Institute of Biological Resources (NIBR no. 2013-02-001) funded by the Ministry of Environment of Korea.]

A023

Lacibacter nakdongensis sp. nov., Isolated from Sediments of Nakdong River

Ji-Hye Han, Kiwoon Baek, and Mi-Hwa Lee*

Bacterial Resources Research Team, Freshwater Bioresources Research Division, Nakdonggang National Institute of Biological Resources (NNIBR)

A Gram-negative, non-spore-forming, orange pigmented bacterium, designated strain SS2-56T was isolated from the sediments of Nakdong River in Sangju-si, Gyeongsangbuk-do. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate SS2-56T belongs to the family Chitinophagaceae, and was most closely related to the type strains of Lacibacter daechungensis H32-4T (96.64%) and Lacibacter cauensis NJ-8T (96.10%). Growth occurred at 20-37°C (optimum temp. 30°C). The most abundant fatty acids in whole cell of strain SS2-56T were iso-C16:0 (43.3%), iso-C17:0 3-OH (19.1%), summed feature 3 (C16:1 ω7c and/or C16:1 ω6c; 7.5%) and iso-C15:1 G (6.5%). The biochemical analyses using the API kit, Biolog and hydrolysis tests indicated that the strain could be clearly distinguished from related species of Lacibacter. Based on the results of the polyphasic taxonomic analysis, a novel species, Lacibacter nakdongensis sp. nov., is proposed to accommodate this novel isolate. The type strain is SS2-56T.

A026

Emticicia fontis sp. nov., Isolated from Freshwater

Gi Gyun Nam, Yochan Joung, and Jang-Cheon Cho*

Department of Biological Sciences, Inha University

A bacterial strain, designated IMCC1731T, was isolated from freshwater in Korea and characterized using a polyphasic taxonomic approach. Growth occurred at 10-37°C (optimum 25°C) and with 0-1% NaCl. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain IMCC1731T

belonged to the genus Emticicia within the family Cytophagaceae and was most closely related to Emticicia ginsengisoli Gsoil 085T (98.11% sequence similarity) and Emticicia oligotrophica DSM 17448T (94.67%). The DNA-DNA relatedness of strain IMCC1731T with respect to Emticicia ginsengisoli Gsoil 085T was less than 70%. The G+C content of strain IMCC1731T was 37.82 mol%. The predominant cellular fatty acids were summed feature 3 (C16:1 ω6c and/or C16:1 ω7c) and iso-C15:0. Based on the physiological, chemotaxonomic characteristics, DNA-DNA hybridization relatedness and 16S rRNA genes analysis, stain IMCC1731T is considered to represent a novel species of Emticicia, for which Emticicia fontis sp. nov. is proposed. The type strain is IMCC1731T.

Poster

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A027

Genomic Characteristics of Strain IMCC26207, a Non-colony-forming Actinobacterium, Isolated from an Oligotrophic Freshwater Lake

Suhyun Kim, Ilnam Kang, and Jang-Cheon Cho*


Although the phylum Actinobacteria is a common and numerically important component in freshwater habitats, their potential roles in biogeochemical process remain unknown due to the scarcity of reference genomic resources. We report here the draft genome sequence of strain IMCC26207, a non- colony forming freshwater actinobacterium, with the description of the genome properties. Strain IMCC26207 was isolated from the surface layer of Lake Soyang by the dilution-to-extinction culturing method. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain IMCC26207 formed a distinct lineage in the order Acidimicrobiales of the phylum Actinobacteria. The closest relative among previously identified bacteria was ‘Candidatus Microthrix parvicella’ (16S rRNA similarity: 91.7%). The draft genome consisted of 10 contigs with a total size of 3.3 Mb and an average G+C content of 57.3%. The IMCC26207 genome was predicted to contain 2,975 protein-coding genes and 51 RNA genes, including 45 tRNA genes. Approximately 76% of the protein coding genes could be assigned with a specific function. Annotation of the IMCC26207 genome showed several traits of adaptation to living in oligotrophic freshwater environments. Comparative analysis revealed that the IMCC26207 genome was distinct from ‘Candidatus Microthrix’ genomes; therefore, we propose the name ‘Candidatus Limnosphaera aquatica’ for this bacterium. [Supported by a grant of the Mid-Career Research Program through the NRF]

A028

Isolation of Three New Flavobacteriaceae Strains, Their Genome Characteristics, and Proposal of the Name Aurantibacter yeongjongensis gen. nov., sp. nov

Yeonjung Lim1, Yochan Joung1, Seung-Jo Yang2, and Jang-Cheon Cho1*1Department of Biological Sciences, Inha University, 2National Marine Biodiversity Institute of Korea

Three Gram-stain-negative, non-motile, orange-colored bacterium, designated IMCC2192T, IMCC2184, and IMCC2644 were isolated from a marine solar saltern. Cellular growth occurred at 25-30℃, pH 6.0-7.5, and with 1-4% (w/v) NaCl. The DNA G+C content of the strains were 35.9-36.0 mol%. The major respiratory quinone was menaquionone-6 (MK-6) and predominant cellular fatty acids were iso-C15:1 G, iso-C15:0 and iso-C17:0 3OH. Major polar lipids were aminolipids, phospholipids, phosphatidylethanolamine. Phylogenetic analysis based on 16S rRNA gene sequence indicated that the strains belonged to the family Flavobacteriaceae and formed a distinct lineage that was independent of closely related genera: Winogradskyella (95.1-92.8%), Flavirhabdus (94.2-94.3%), Bizionia (92.8-95.1%), and Corallibacter (93.9-94.0%). Average nucleotide identity (ANI) values (>98%) based on whole genome sequence data (IMCC2192T and IMCC2644) and DNA-DNA relatedness (>90%) based on hybridization (IMCC2192T and IMCC2184) indicated that the strains belong to the same genomics species. The genome size of strains IMCC2192T and IMCC2644 were 2.9 Mb and 2.8 Mb, respectively, and shared general genomic properties of the Flavobacteriaceae. In this regard, the strains are considered to represent a novel genus, for which Aurantibacter yeongjongensis gen. nov., sp. nov is proposed. The type strain is IMCC2192T (=KACC18774T). [Supported by a grant from the Marine Biotechnology Program (PJT200620) funded by the MOF, Korea]

A029

Flavobacterium inkyungensis sp. nov., Isolated from Freshwater of an Artificial Pond

Miri Park, Yochan Joung, Gi Gyun Nam, and Jang-Cheon Cho*


A Gram-staining-negative, non-motile, yellow pigmented bacterium, designated strain YMO21T, was isolated from an artificial pond (Inkyung Lake), Incheon. Growth of strain YMO21T occurred at 4-37°C (optimum, 30°C), at pH 7.0-8.0 (optimum, pH 7.0) and with 0-0.25% NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YMO21T belonged to the genus Flavobacterium and most closely related to Flavobacterium columnare IFO 15943T (97.8 % sequence similarity), Flavobacterium terrae R2A1-13T (97.2 %) and Flavobacterium vireti THG-SM1T (96.7%). DNA G+C content of strain YMO21T was 32.1 mol%. The major fatty acids were iso-C15:0 (31.1%), iso-C15:1 G (17.6%), iso-C15:0 3OH (9.5%) and the major isoprenoid quinone was menaquinone-6 (MK-6). The major polar lipid of strain YM021T was phosphatidylethanolamine (PE). Strain YM021T showed low DNA–DNA relatedness with Flavobacterium columnare IFO 15943T (62.4 %). Based on the analysis of 16S rRNA gene sequences and biochemical characterization, strain YMO21T was considered to represent a novel species in the genus Flavobacterium, for which the name Flavobacterium inkyungensis sp. nov. is proposed. [Supported by grants from the Mid-Career Research Program through the National Research Foundation (NRF) funded by the Ministry of Science, ICT, and Future Planning]

A030

Spirosoma aerophilum sp. nov., Isolated from Air Sample

Soo-Jin Kim, Jae-Hyung Ahn, Hang-Yeon Weon, Seung-Beom Hong,Soon-Ja Seok, Jeong-Seon Kim, and Soon-Wo Kwon*

Agricultural Microbiology Division, National Institute of Agricultural Science, Rural Development Administration

A rod-shaped, yellow-colored, Gram-stain-negative, non-flagellated, aerobic bacterium, designated as 5516J-17T, was isolated from an air sample collected from Jeju Island, Republic of Korea. It grew in the range of 10-37°C (optimum 28-30°C), pH 6.0-11.0 (optimum, pH 7.0) and 0-1% NaCl (w/v). Phylogenetic trees generated using 16S rRNA gene sequences revealed that strain 5516J-17T belonged to the genus Spirosoma and showed 96.9% sequence similarity to the most closely related species, Spirosoma linguale DSM 74T. The cellular fatty acids composed of large amounts (>10% of total fatty acids) of summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and C16:1 ω5c, and moderate amounts (5-10% of total fatty acids) of iso-C17:0 3-OH, iso-C15:0 and C16:0. The DNA G+C content was 55.7 mol% and MK-7 was the predominant isoprenoid quinone. Polar lipids were phosphatidylethanolamine, two unknown aminophospholipids, one unknown aminolipid and one unknown lipid. On the basis of phenotypic and polyphasic taxonomy study, it was suggested that strain 5516J-17T represent a novel species within the genus Spirosoma, with the newly proposed name Spirosoma aerophilum. The type strain is 5516J-17T (=KACC 17323T =DSM 28388T =JCM 19950T).


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A031

Lysobacter terricola sp. nov., Isolated from Greenhouse Soil

Soo-Jin Kim, Jae-Hyung Ahn, Hang-Yeon Weon, Seung-Beom Hong, Soon-Ja Seok, Jeong-Seon Kim, and Soon-Wo Kwon*

Agricultural Microbiology Division, National Institute of Agricultural Science, Rural Development Administration

A strain 5GH22-11T, which was isolated from greenhouse soil in Yangpyeong region, Gyeonggi province, Republic of Korea, was characterized as aerobic, Gram-stain-negative, flagellated, rod-shaped bacterium. The 16S rRNA gene sequence analysis showed that strain 5GH18-14T showed the highest sequence similarities with Lysobacter niabensis GH34-4T (98.6%), Lysobacter yangpyeongensis GH19-3T (98.1%), Lysobacter fragariae THG-DN8.7T (97.9%), Lysobacter terrae THG-A13T (97.3%), Lysobacter rhizosphaerae THG-DN8.3T (97.2%), Lysobacter tyrosinelyticus THG-DN8.2T (97.2%) and Lysobacter oryzae YC6269T (97.2%), revealing less than 95.5% sequence similarities with all the other validly published species. Predominant quionone of strain 5GH18-14T was Q-8. Polar lipids were large amounts of phosphati-dylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and phos-phatidylmonomethylethanolamine, and moderate or small amounts of three unknown phospholipids and two unknown aminophospholipids. DNA-DNA hybridization values with the closely related species were below 70%. DNA G+C content is 65.9 mol%. Based on the phylogenetic, physiological and chemotaxonomic data, it was demonstrated that strain 5GH18-14T represents a novel species of the genus Lysobacter, for which the name Lysobacter terricola sp. nov. is proposed. The type strain is 5GH18-14T (KACC 16954T =JCM 30862T).

A032

Martelella suaedae sp. nov. and Martelella limoniae sp. nov., Isolated from the Root of Halophytes

Eu Jin Chung1,2, Jung Moon Hwang1,2, Kyung Hyun Kim3, Che Ok Jeon3, and Young Ryun Chung1*1Division of Applied Life Science (BK21 Plus), Plant Molecular Biology & Biotechnology, 2Freshwater Bioresources Culture Research Division, Nakdonggang National Institute of Biological Resources, 3Department of Life Science, Chung-Ang University

Two Gram-negative, aerobic and endophytic bacterial strains, designated YC7033T and YC7034T, were isolated from the roots of halophytes (Suaeda maritime and Limonium tetragonum) inhabiting in tidal flats of Namhae Island, Korea, respectively. Strains YC7033T and YC7034T grew at 10–40°C (optimum at 28–30°C) and 4–40ºC (optimum at 28–30°C), respectively. Strains YC7033T and YC7034T were able to grow at NaCl concentrations of 1–9% and 1–6%, respectively. The phylogenetic analyses of 16S rRNA gene sequences showed that two strains were closely related to Martelella endophytica YC6887T, Martelella mangrovi BM9-1T, Martelella radicis BM5-7T and Martelella mediterranea DSM 17316T with 97.6–99.1% similarities in 16S rRNA gene sequences. Sequence similarities with the type strains of another closely related genus, Rhizobium, were lower than 95.0%. Both strains contained ubiquinone-10 (Q-10) as the major respiratory quinone system. The G+C contents of the genomic DNA of strains YC7033T and YC7034T were 52.8 mol% and 62.2 mol%, respectively. The major fatty acids of both strains YC7033T and YC7034T were summed feature 8 (C18:1ω7c and/or C18:1 ω6c) and C19:0 cyclo ω8c. On the basis of phylogenetic analysis, biochemical and phenotypic characteristics, strains YC7033T and YC7034T represent two novel species of the genus Martelella and we propose the names Martelella suaedae sp. nov.

A033

Lacinutrix chionocetis sp. nov., Isolated from Gut of a Red Snow Crab

Hyangmi Kim1, Sunjoo Park2, Hyun-Woo Oh3, Kyung Sook Bae2, and Doo-Sang Park2,4*1Freshwater Bioresources Culture Research Division, Nakdonggang National Institute of Biological Resources, 2Bicrobiological Resources Center, KRIBB

3Core Facility Management Center, KRIBB, 4Department of Green Chemistry and Environmental Biotechnology, UST

A Gram-negative, strictly aerobic, non-motile and rod-shaped bacterial strain, designated MAB-07T, was isolated from gut of a red snow crab. The novel strain grew optimally at 25°C, at pH 7.0–8.0 and in the presence of 3% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain MAB-07T belongs to the type strains of species of the genus Lacinutrix. Strain MAB-07T exhibited 16S rRNA gene sequence similarity values of 95.5-97.8% to the type strains of species of the genus Lacinutrix. The predominant cellular fatty acids of strain MAB-07T were iso-C15:1 G (27.5%) and iso-C15:0 (21.7%). The major respiratory quinine was identified as MK-6. The polar lipids consisted of phosphatidylethanolamine, two unidentified phospholipids four unidentified aminolipids. The genomic DNA G+C content was determined to be 33.5% and its DNA-DNA relatedness values with the type strains of Lacinutrix venerupis, Lacinutrix mariniflava, Lacinutrix jangbogonensis and Lacinutrix algicola were 28–35%. Based on the data from this polyphasic taxonomic study, strain MAB-07T is considered to represent a novel species of the genus Lacinutrix, for which the name Lacinutrix chionocetis sp. nov. is proposed. The type strain is MAB-07T (=KCTC 42767T).

A034

Genomic Characterization of Strain IMCC26134, a Freshwater Verrucomicrobial Strain Isolated from Lakewater

Ahyoung Choi1,2, Ilnam Kang1, Suhyun Kim1, and Jang-Cheon Cho1*1Department of Biological Sciences, Inha University, 2Culture Techniques Research Team, Nakdonggang National Institute of Biological Resources

Strain IMCC26134, a freshwater verrucomicrobial strain, was isolated from Lake Soyang by dilution-to-extinction culturing and formed small colonies on semisolid agar. IMCC26134 is an aerobic microorganism with optimal growth at pH 7.0, at 20°C and salinity ≤ 0.2% NaCl. In phylogenetic analyses, strain IMCC26134 constituted a robust branch within the family Opitutaceae of the phylum Verrucomicrobia. The closest relative of this strain was ‘Candidatus Diplosphaera colotermitum’ with a 16S rRNA gene sequences similarity of 92.6%. Here we describe the features of strain IMCC26134, together with the complete genome sequence information and its annotation. The complete genome was comprised of one circular chromosome, with a length of 4,001,000 bp and a G+C content of 61.3%. The IMCC26134 genome was predicated to contain 3,050 protein-coding genes and 56 genes for RNAs. The majority of the protein coding genes (75.3%) were assigned a putative function. The genome of strain IMCC26134 revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, polysaccharide lyases and carbohydrate esterase, confirming that this organism was well equipped for the hydrolysis of diverse polysaccharides. Strain IMCC26134 genome enhance understanding of the poorly known freshwater Verrucomicrobia phylum.

Poster

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A035

Oleiagrimonas sediminis sp. nov., a Marine Bacterium Isolated from Tidal Flat Sediment and Emended Description of the Genus Oleiagrimonas Fang et al. 2015 and Oleiagrimonas soli

Sung-Hyun Yang1, Hyun-Seok Seo1, Chi Nam Seong2, and Kae Kyoung Kwon1*1Marine Biotechnology Research Center, Korea Institute of Ocean Science & Technology, 2Department of Biology, College of Life Science and Natural Resources, Sunchon National University

A Gram-negative, aerobic, rod-shaped (1.4–3.6 ㎛ × 0.4–0.6 ㎛) and motile marine bacterium, designated as MEBiC09124T was isolated from tidal flat sediment of the Sun cheon province, South Korea. The 16S rRNA gene sequence analysis revealed that strain MEBiC08749T showed high similarity with the Oleiagrimonas soli 3.5XT (96.7%). Growth was observed at 18–38°C (optimum 30°C), at pH 4.0–8.5 (optimum pH 7.5) and with 0–6% (optimum 2.5%) NaCl. The predominant cellular fatty acids were iso-C15:0 (22.6%), iso-C16:0 (16.0%), iso-C17:0 (10.0%) and summed feature 9 (comprised of 10-methyl-C16:0 and/or iso-C17:1ω9c; 22.5%). The DNA G+C contents is 66.1 mol%. The major respiratory quinone is Q-8. Several phenotypic characteristics such as production of acetoin and Enzyme activities of Trypsin, α-chymotrypsin and N-acetyl-β-glucosaminidase differentiate strain MEBiC09124T from O. soli 3.5XT. On the basis of this polyphasic taxonomic data, strain MEBiC09124T should be classified as a novel species in the genus Oleiagrimonas and it is proposed as Oleiagrimonas sediminis sp. nov. The type strain is MEBiC09124T (=KCCM 43131T =JCM 30904T). Emended descriptions of the genus Oleiagrimonas Fang et al. 2015 and Oleiagrimonas soli are also given. [Supported grants from KIOST & MBRB.]

A036

Perlucidibaca aquatica sp. nov. Isolated from Freshwater

Kiwoon Baek, Ji-Hye Han, and Mi-Hwa Lee*

Nakdonggang National Institute of Biological Resources

A Gram-staining-negative, non-motile, non-pigment, aerobic and rod-shape bacterium, designated BK-297T was isolated from small valley of the Samcheok, Korea. Optimal growth of strain BK-297T was observed at 30°C, pH 7.0 and in the presence of 0.5% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BK-297T belonged to the genus Perlucidibaca, forming a robust clade with member of the genus, and was most closely related to Perlucidibaca piscinae (97.8% similarity). The major fatty acids were C16:1 ω7c and/or C16:1 ω6c (25.6%) and C16:0 (14.1%). On the basis of phyogentic analysis and genetic data, strain BK-297T represents a novel species of the genus Perlucidibaca, for which the name Perlucidibaca aquatica sp. nov. is proposed.

A037

Palleronia salinaecis sp. nov., a Halophilic Species Isolated from Solar Saltern

Suhk Hwan Park and Geon Hyoung Lee*

Department of Biology, Kunsan National University

A novel Gram-negative, strictly aerobic bacterial strain was isolated from solar saltern. This bacterial strain, designated as Kmb80T, was halophilic, non-motile, non-spore forming, and rod-shaped. The colonies were 0.8-1 mm in diameter, circular, pale-pink pigmented and with smooth edge. The strain was positive for oxidase and catalase activities. Growth occurred at pH 6.0–9.5 with optimal growth at pH 7.0, at temperatures of 20–40°C with optimal growth at 30°C, and in the presence of 0.5–20% (w/v) NaCl with optimal growth at 5% (w/v). Based on the 16S rRNA gene sequence analysis, strain Kmb80T was assigned to the genus Palleronia within the family Rhodobacteraceae and was closely related to the type strain of Palleronia soli with 96.9% of 16S rRNA gene sequence similarity. The major cellular fatty acids of strain Kmb80T were C18:1 ω7c and/or C18:1 ω6c (38.8%) and C 19:0 cyclo ω8c (30.4%). Ubiquinone 10 (U-10) was found to be the major respiratory quinone. The G+C content of the genomic DNA was 67.3 mol%. On the based phenotypic properties clearly separated the strain from other species of the genus. Accordingly, a new species of the genus Pallronia, Pallronia salinaecis sp. nov., is proposed (type strain =Kmb80T =KCTC32522T = JCM19390T).

A038

Analysis of Non-ribosomal Peptide Synthetase Gene Cluster for Tolaasin Biosynthesis

DoWon Kang1,2, JungHun Jeon1,2, Chang-Won Lee1,2, and Jae Won Kim1,2*1Division of Applied Life Science (BK21 Research), 2Research Institute of Life Sciences, Gyeongsang National University

The whole sequence of non-ribosomal peptide synthetase(NRPS) gene cluster was analysed. The gene cluster was composed of adenylation domains, condensation domains, and thiolation domains for 18 amino acids, consisting of tolassin, respectively. One thioesterase domain was found in terminal region. Analysis of non-ribosomal peptide synthetase gene cluster for tolassin biosynthesis. When the whole sequence of NRPS was compared to Pseudomonas tolaasii 6264 and Pseudomonas tolaasii NCPPB 2192, there was not strong homology with these gene sequences from database. Lux R region was present in up-stream region, therefore NRPS should be controlled by Lux R type regulation. Although gene sequence for NRPS from Pseudomonas tolaasii PMS 117 was not completed, the homology between two strains was shown 89% homology in DNA sequence.


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A039

The Diagnostic Characteristics are Highly Homoplasious Used in Cladonia gracilis and Cladonia cornuta

Jae Eun So1,2, Soon Gyu Hong1,2, and Ji Hee Kim1*1Division of Polar Life Sciences, Korea Polar Research Institute2Department of Polar Sciences, University of Science & Technology

The genus Cladonia usually lives on soil, moss and dead tree, and more than 400 species hitherto have been documented. Traditionally, the genus was divided into 7 taxonomic sections by morphology. However, the taxonomical division is not well accord with that of molecular phylogenetic research. The genus has a high morphological diversity, and the delimitation of many species has remained unresolved. The Cladonia gracilis group and allies in the section Cladonia shows an extreme phenotypic variety. Cladonia gracilis and Cladonia cornuta which are member of the C. gracilis group have been identified by variable characteristics. The present work deals with the taxonomic problem between C. gracilis and C. cornuta by confirming the consistent phenotypic characteristics again with molecular phylogeny in species level. About 60 specimen were analyzed by 36 morphological and chemical characteristics using the DNA sequences with three loci (ITS rDNA, mtSSU and LSU rDNA). Specimen were collected from a few different regions covering bipolar areas and Korea. Phenotypic data were analyzed with several statistical methods. The study indicates that the consistent clade was not constructed in these species among the species level. The results show the unclear distinction between two species for secondary metabolites and geographical distribution. The characteristics used for classical diagnosis may be highly homoplasious.

A040

Zygomycete Fungi from Animal Dung in Korea

Thi Thuong Thuong Nguyen, Seo Hee Lee, Sarah Bae, Sun Jeong Jeon, and Hyang Burm Lee*

Division of Food Technology, Biotechnology and Agrochemistry, College of Agriculture & Life Sciences, Chonnam National University

During a survey of zygomycota biodiversity in Korea, six isolates EML-DG8-1, EML-DG-NH3-1, EML-UKD2-1, EML-UKD9-1, EML-UKD5-1, EML-UKD7-1 were isolated from animal dung samples from June 2014 to Ferbuary 2016. Sequence analysis by BLASTn search indicated that the isolates, EML-DG8-1, EML-DG-NH3-1, EML-UKD2-1, EML-UKD9-1, EML-UKD5-1, EML-UKD7-1 were closets to Absidia sp. (GenBank accession no. KC007314), Absidia glauca (GenBank accession no. AY944875), Mucor genevensis (GenBank accession no. JN206044), Mucor hiemalis (GenBank accession no. JX045814), Mucor circinelloides (GenBank accession no. KT876701), and Mucor mucedo (GenBank accession no. EU484199) with identity values of 93.9% (539/574 bp), 97.7% (606/620 bp), 99.5% (370/372 bp), 99.1% (568/573 bp), 99.1% (539/544 bp), and 99.8% (645/646 bp), respectively. Based on the morphological characteristics and multigene phylogenetic analyses, the EML-DG8-1 isolate was identified as a new species, namely, Absidia stercoraria sp. nov. EML-DG8-1. The EML-DG-NH3-1 and EML-UKD2-1 isolates were identified as an unrecorded species: A. glauca and M. genevensis, respectively. The EML-UKD9-1, EML-UKD5-1 and EML-UKD7-1 isolates were identified as M. hiemalis, M. circinelloides, and M. mucedo, respectively in Korea

A041

Chthonobacter albigriseus gen. nov., sp. nov., Isolated from Grass-field Soil in Korea

Do Hak Kim, Minsun Kim, Hansol Kim, Keunsoo Kang, and Tae-Young Ahn*

Department of Microbiology, College of Natural Sciences, Dankook University

ED7T―a Gram-negative, non-motile, non-gliding, and aerobic bacterial strain―was isolated from grass-field soil in Cheonan, Korea. Strain ED7T was able to grow at 20–42°C (optimum at 30–35°C), at pH 7.0–8.5 (optimum at pH 7.5–8.0), and at the absence of NaCl. According to similarities of 16S rRNA gene sequences, strain ED7T was most closely related to the genera Labrenzia (≤93.3%), Pleomorphomonas (≤93.1%), and Prosthecomicrobium (≤93.1%). Phylogenetic analysis based on 16S rRNA gene sequence of strain ED7T revealed that it was affiliated with the order Rhizobiales, being most closely related to the genus Pleomorphomonas. Strain ED7T did not contain the nifH gene while its closest relatives, Pleomorphomonas koreensis, P. oryzae, and Prostehcomicrobium pneumaticum did. The DNA G+C content of the genomic DNA was 71.7 mol%. The predominant fatty acids of strain ED7T were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), summed feature 2 (unknown fatty acid of 10.9525, C12:0 aldehyde, C14:0 3-OH and/or C16:1 iso I), C18:1 ω7c 11-methyl1, and C16:0 3-OH. The major isoprenoid quinone was ubiquinone 10 (Q-10). Based on phenotypic, chemotaxonomic and genotypic characteristic data, the strain ED7T could be differentiated from other genus and considered that ED7T strain represents a novel species of a new genus in the order Rhizobiales, for which the name Chthonobacter albigriseus gen. nov., sp. nov. is proposed. The type strain is ED7T (=JCM 30603T =KCTC 42450T).

A042

Characterization and Description of Novel Strain RP18T, Isolated from the Forest Soil

Yongseok Ko, Han a Cho, Seoyoun Koo, and Tae-young Ahn*

Department of Microbiology, College of Natural Sciences, Dankook University

An aerobic, Gram negative, yellow-pigmented bacterium, strain RP18T was isolated from the forest soil, Gwangju, Korea. The strain grew optimally on R2A medium at 30°C and in the absence of NaCl. The phylogenetic analysis based on 16S rRNA gene sequence suggested that the strain RP18T belonings to the genus Sphingomonas, exhibiting the highest level of sequence similarity with type strain of Sphingomonas kyeonggiense (95.96%), Sphingomonas pituitosa (95.37%) and Sphingomonas jejuensis (94.71%). The major fatty acids of strain RP18T were, C14:0 2-OH,anteiso- C15:0, C16:0, C17:0 w6c, C18:1 w7c 11-methyl (>3% of the total fatty acids). Based on the evidence, The strain RP18T must be classified as a new species of the genus Sphingomonas.

Poster

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A043

Seddomonas intestinalis gen. nov., sp. nov., Isolated from Human Faeces

Boram Seo1, Ju Eun Yoo1, Yung Mi Lee3, and GwangPyo Ko1,2*1Department of Environmental Health, Seoul National University, 2Center for Human and Environmental Microbiome, Seoul National University, 3Division of Polar Life Sciences, Korea Polar Research Institute

A strictly anaerobic Gram-stain-positive, non-motile and non-spore-forming bacterial strain, designated BR31T, was isolated from a fecal sample of a healthy human. Bacterial cells were ivory-colored rods with rounded ends and approximately 1.4-2.1 x 0.5-0.6 μm in size. According to the comparative analysis based on 16S rRNA gene sequence, strain BR31T formed a distinct phyletic lineage that reflects a novel genus within the Clostridium cluster XIVa in the family Lachnospiraceae, with the highest similarity to the Eubacterium contortum DSM 3982T (94.6%). The DNA G+C content was calculated to be 47.0 mol% from whole-genome sequence. There were predicted genes associated with synthesis of teichuronic acid, polyamines, diaminopimelic acid and polar lipids. The major metabolic end product of glucose was acetic acid, which was in line with those of most members of the family. However, the profile of major cellular fatty acids (C16:0, C14:0, summed feature 4 and C13:0) and overall enzyme activity demonstrated the phenotypic differentiation of strain BR31T from other closely related genera. Thus we suggest that strain BR31T represents a novel genus and species, for which the name Seddomonas intestinalis gen. nov., sp. nov. is proposed. The type strain is BR31T (=KCTC 15482T =JCM 30748T).[This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (NRF-2015R1A2A 1A10054078). B. Seo was supported by the Global Ph.D. Fellowship program, NRF Grant funded by the Korean Government (NRF-2012H1A2A1049234).]


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B001

Occurrence of Viable, Red-pigmented Haloarchaea in the Plumage of Captive Flamingoes

Seong Woon Roh and Jong-Soon Choi*

Biological Disaster Analysis Group, Korea Basic Science Institute

Flamingoes (Phoenicopterus spp.) whose plumage displays elegant colors, inhabit warm regions close to the ocean throughout the world. The pink or reddish color of their plumage originates from carotenoids ingested from carotenoid-abundant food sources, since flamingoes are unable to synthesize these compounds de novo. In this study, viable red-colored archaeal strains classified as extremely halophilic archaea (i.e., haloarchaea) and belonging to the genera Halococcus and Halogeometricum were isolated from the plumage of flamingoes in captivity. Detailed analysis for haloarchaeal community structure in flamingo feathers based on metagenomic data identified several haloarchaeal genera and unclassified sequences of the class Halobacteria at the genus level. Carotenoid pigment analyses showed that a bacterioruberin precursor carotenoid in haloarchaea was identical to one of the pigments found in flamingo plumage. To the best of our knowledge, this is the first report of viable extremophilic archaea in avian plumage, thus contributing to our understanding of the ecology of haloarchaea. The potential influence of haloarchaea as an environmental factor determining avian plumage coloration should be investigated in further studies.

B002

Diversity and Distribution of Nitrite Oxidoreductase Alpha Subunit (nxrA) Gene in the Marine Sediments

Rani Sundas, Hyeon-Woo Koh, and Soo-Je Park*

Department of Biology, Jeju National University

Nitrite-oxidizing bacteria (NOB) are chemolithoautotrophs catalyzing the oxidation of nitrite to nitrate, which is the second step of aerobic nitrification. In marine systems, Nitrospina are major member as contribute to nitrogen cycle. Still, two strains of the genus Nitrospina have been only isolated from marine environment. Considering on their ecological contribution, the investigations for their ecophysiology and environmental distribution have been understudied due to fastidious cultivation and the insufficiency of a functional gene marker. To estimate abundance, diversity and distribution of Nitrospina, here we introduce nxrAencoding the alpha subunit of nitrite oxidoreductase which used as functional and phylogenetic marker for that, and successfully applied into several marine sediments geographically distant location. We observed that Nitrospina diversities of the both polar sediments were significantly lower than that of the non-polar samples. Moreover, nxrA-like sequences revealed an unexpected microdiversity of Nitrospina with about 15000 identified operational taxonomic units from six marine sediments. The nxrA gene copy numbers were ranged to 6.8 × 104 per gram of marine sediment sample. Conclusion, this study estimated the distribution and diversity of Nitrospina and provided fundamental information on the potential contribution to nitrogen cycle in marine sediments.[Supported by grants from NRF (2015R1C1A1A01053750 & 2015M3D-3A1A01064881)]

B003

Comparative Analysis of Microbial Communities and Soil Organic Carbon Utilization Associated with the Depth and Thawing Effects on Tundra Soil in Alaska

Ha Ju Park1, Hyun Park1, Bang Yong Lee2, Yoo Kyung Lee2, and Dockyu Kim1*1Division of Life Sciences, 2Arctic Research Center, Korea Polar Research Institute

In high-latitude regions, temperature has risen twice as fast as the global average (0.3°C per decade) and this leads to the increase in microbial degradability against soil organic carbon (SOC). Furthermore, the decomposed SOC is converted into green-house gases and their release could further increase the rate of climate change. Thus, understanding the microbial diversity and their functions linked with SOC degradation in soil-thawing model is necessary. In this study, we divided SOC-rich soil from Alaska into two depth regions (30−40 cm and 50−60 cm of depth) and incubated that for 108 days at 0°C. A total of 111,804 reads were obtained through metagenomic study during the microcosm experiments, and 574−1,128 of bacterial operational taxonomic units (OTUs) and 30−57 of archaeal OTUs were observed. Taxonomic analysis showed that the distribution of bacterial taxa was significantly different between two samples, while archaea was similar. In detail, the relative abundance of phyla Actinobacteria and Firmicutes largely increased in 30−40 cm and 50−60 cm of soil depths, respectively. Weight measurement and gel permeation chromatography of the SOC extracts demonstrated that polymerization of humic acids, main component of SOC, occurred during the microcosm experiments. Taken together our results indicate that these two bacterial phyla could play a key function in SOC degradation and utilization in cold tundra soil. [This work was supported by KOPRI (PE16070).]

B004

Metagenomic and Functional Analyses of the Consequences of Reduction of Bacterial Diversity on Soil Functions and Bioremediation in Diesel-contaminated Microcosms

Jaejoon Jung1, Laurent Philippot2, and Woojun Park1*1Department of Environmental Science and Ecological Engineering, Korea University2INRA Dijon, UMR 1347 Agroecologie, Dijon, France

The relationship between microbial biodiversity and soil function is an important issue in ecology, yet most studies have been performed in pristine ecosystem. Here, we assess the role of microbial diversity in ecological function and remediation strategies in diesel-contaminated soils. Soil microbial diversity was manipulated using a removal by dilution approach and microbial functions were determined using both metagenomic analyses and enzymatic assays. A shift from Proteobacteria- to Actinobacteria- dominant communities was observed when species diversity was reduced. Metagenomic analysis showed that a large proportion of functional gene categories were significantly altered by the reduction in biodiversity. The abundance of genes related to the nitrogen cycle was significantly reduced in the low-diversity community, impairing denitrification. In contrast, the efficiency of diesel biodegradation was increased in the low-diversity community and was further enhanced by addition of red clay as a stimulating agent. Our results suggest that the relationship between microbial diversity and ecological function involves trade-offs among ecological processes, and should not be generalized as a positive, neutral, or negative relationship.

Poster

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B005

Effect of Oil Contamination on the Resilience of Taxonomic and Functional Structure in the Korean Tidal Flat

Jaejin Lee1, Boram Kang2, and Tae Kwon Lee2*1Division of Life Sciences, Korea Polar Research Institute2Department of Environmental Engineering, Yonsei University

A significant portion of crude oil from the oil shipping in the West Sea, Korea was transported to the tidal flat, where is an important habitat or ecological zone due to high biological diversity. The objectives of this study were (i) to identify and characterize oil degrading bacteria, ii) to characterize the recovery of taxonomic and functional structures over time in Korean tidal flat. This study was conducted at Uihang-ri area, Taean, where chemical analysis revealed that total petroleum hydrocarbon (C12 to C34) decreased from 3 μg/g to 0.5 μg/g over one year. Cycloclasticus, Alcalivorax, Pseudomonas and Thalassolituus were the predominant members as oil degrading microorganisms during the crude oil degradation. Taxonomic (16S rRNA gene) and functional (aromatic ring hydroxylating dioxygenase) community analysis revealed a distinct response to oil contamination by depth, but the initial community structure was recovered in both approches over one year. The combination of 16S rRNA and dioxygenase gene analyses resulted in better understanding of the contributions to the crude oil removal of the indigenous microbial community and high degree of resilience of microbial community and diversity after the contamination. [Supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2015R1D1A1A02061084)]

B006

Bacterial Diversity and Species Composition in Three Endemic Baikalian Sponges

Eun-Young Seo1, Dawoon Jung1, Yochan Joung2, and Tae Seok Ahn1*1Department of Environmental Science, Kangwon National University2Department of Biological Sciences, Inha University

In Baikal, Russia, 3 species of sponges are living. Sponges are unique organisms with many species harboring symbiotic microbes that produce novel bioactive compounds. To investigate bacterial diversity of Baikalian sponges, specimens of Lubomirskia baicalensis, Baikalospongia intermedia and Swartschewskia papyracea collected from Lake Baikal were processed by pyrosequencing. We found differences in the species composition and diversity in bacteria among these sponges. Cyanobacteria accounted for the highest proportion and second group was Proteobacteria in three sponges. The bacterial communities in B. intermedia and L. baicalensis were highly similar but less similar from the bacterial community associated with S. papyracea. Diversity of the bacterial community in S. papyracea was higher than in L. baicalensis and B. intermedia. Especially, the relative abundance of Actinobacteria was higher in S. papyracea. Bacterial species in phyla Acidobacteria and Gemmatimonadetes were only found in S. papyracea.

B007

Cecal Microbiome from Broiler Chickens Differing in Body Weight and Sex

Kyu Chan Lee1, Dong Yong Kil2, and Woo Jun Sul1*1Department of Systems Biotechnology, Chung-Ang University2Department of Animal Science and Technology, Chung-Ang University

Microbial community in ceca is involved in several important metabolisms such as water regulation, energy production during digestion and carbohydrate fermentations. To view the differences of microbial communities in the ceca, we analyzed chicken cecal microbiota based on bodyweights (high, medium, low) and sex. In this study, 2×3 factorial experiment was designed to predict bacteria positively related to host genotype and BW, using Linear Discriminant Analysis Effect Size (LEfSe). Bodyweights and sex groups were clearly separated and statistically significant (p-value < 0.05). In general, Actinomycetales members and Centipeda were positively related to low BW while Enterococcus, unclassified Lactobacillales, unclassified IncertaeSedis XIII and unclassified Ruminococcaceae were related to high BW. In female chickens, several Firmicutes members (Sporobacter, Papillibacter, Unclassified Firmicutes) were mainly observed, positively relating to high and low BW. In male chickens, on the other hand, many Firmicutes members such as Coprococcus, Dorea and Roseburia were related to high BW rather than to medium and low BW. Since bacterial diversities abided by BW and sex significantly differed, understanding of bacterial communities deciphering chicken BW and health will help to improve breeding of chickens in poultry industries.

B008

Structural Analysis of the Sensory Domain of the Aromatic-responsive Transcriptional Activator PoxR from Ralstonia eutropha

Vinod Vikas Patil1,2, Kwang-hyun Park1,2, Seung Goo Lee1,2, and Eui-Jeon Woo1,2*1Korea Research Institute of Bioscience and Biotechnology, 2University of Science and Technology

Phenol are considered as hazardous pollutants that pose threat to human life and surrounding ecosystem. Some bacteria are known to utilize phenols as a carbon and energy source. PoxR is a transcriptional activator that control the metabolism of phenols. The sensory domain bind phenols and relays the signal to adjacent ATPase and DNA binding domains making it transcriptionally active that leads to transcription of several genes responsible for catabolism of phenol. Thus, it acts as a switch that can control the expression of genes involved in catabolism of phenols. The structural information about the sensory domain has been elusive so far. We have addressed the fundamental question on “How phenols are recognized inside bacterial cell” We coupled X-ray crystallography with isothermal titration calorimetry (ITC) to determine the structure of sensory domain and understand protein ligand interactions. It was observed that the sensory domain of PoxR exist as a tightly bound homodimer with a hydrophobic phenol binding pocket buried deep inside. Each monomer has one zinc binding site. The sensory domain tightly binds to phenol with Kd=1µM as determined by ITC. This study could aid engineering of aromatic responsive proteins for 1) Development of highly sensitive biosensors for detection of diverse aromatic compounds 2) Development of efficient bacterial strains for bioremediation of aromatic compounds.


14 | www.msk.or.kr

B009

Effect of Indigenous Microbiome in Soil on Salmonella enterica Survival

Suin Yang, Sora Kim, Jeong-A Lim, Jin-Woo Park, Jae-Gee Ryu, and Kyu Seok Jung*

Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration

The objective of this study is investigation of the survival of Salmonella enterica, one of the foodborne pathogenic bacteria, in soil which has indigenous microbial community. To do this when Salmonella enterica ATCC 13311 was introduced to soil where cherry tomato had grown for 10 to 15 years in the green-house, survival of S. enterica were verified. S. enterica was inoculated to the autoclaved and non-autoclaved soil (8 log CFU/ml) and grown in green-house. The cell number of S. enterica inoculated in autoclaved soil was maintained about 7-8 log CFU/ml until 21 days. However, in case of the S. enterica inoculated in non-autoclaved soil the population was sharply reduced after 5 days and finally not detected after 21 days. Thus, it was supposed that a viable organism which could not work by autoclaving inhibit growth of S. enterica. Most culturable bacteria in indigenous microbe were identified as Bacillus species by 16s rRNA sequencing. Compared to autoclaved soil, specified species such as Paenibacillus sp (13.7%), Bacillus firmus (5.9%), and Bacillus pumilus (2.0%) were isolated only in non-autoclaved soil. Thus, it was guessed that these Bacillus species can affect survival of S. enterica. [This work was supported by grants from RDA.]

B010

Genomic Analysis of Confluentimicrobium naphthalenivorans NS6, a New Naphthalene Degrader

HyeIm Jeong, Kyung Hyun kim, and Che Ok Jeon*


Confluentimicrobium naphthalenivorans NS6, which was reported as a new novel naphthalene degrader isolated from a tidal flat (Int. J. Syst. Evol. Microbiol., 2015, 4191-4195) was genome-sequenced for the better understanding of its naphthalene biodegradation properties. The complete genome sequence of strain NS6 was characterized by a circular chromosomal genome of 3.65-Mb with a 64.5% G+C content and three plasmids with 184.7 kb, 157.3 kb, and 156.1 kb sizes. The genome analysis of strain NS6 showed that the genome had many dioxygenases that were probably related to the degradation of aromatic compounds and among them, four operons containing hydroxylating dioxygenase alpha subunit coding gene, a key enzyme in naphthalene degradation, could be candidates responsible for naphthalene degradation and a phylogenetic tree based on Rieske domain sequences of hydroxylase alpha subunits showed that the four dioxygenase alpha subunits were respectively clustered into four different clades. To investigate substrate specificities of the four putative naphthalene dioxygenase genes, four dioxygenase alpha subunit-encoding genes were knock-outed using a double cross-over recombination approach and the biodegradation abilities of the resulting knock-out mutants were tested against various aromatic hydrocarbons. Eventually, we will find a gene operon responsible of complete naphthalene-degradation and we will discuss these in more details in the poster section.

B011

Chromobacterium piscinae and Predation by Bdellovibrio bacteriovorus HD100

Wonsic Mun, Seong Yeol Choi, and Robert J. Mitchell*

School of Life Science, Ulsan National Institute of Science and Technology

Bdellovibrio bacteriovorus HD100 is a predatory bacterium that attacks a wide range of Gram-negative bacterial pathogens. In this study, we found that Chromobacterium piscinae, a Gram-negative and violet-pigmented bacterium, is not predated by B. bacteriovorus when cultivated in Dilute Nutrient Broth (DNB). However, C. piscinae can be predated on within a nutrient-free buffer, HEPES. We examined and found that spent-DNB media of C. piscinae significantly delays predation of E. coli MG1655 by B. bacteriovorus HD100. Furthermore, the expression of the vioABCDE genes within E. coli delayed predation of this prey. [Supported by grants from DARPA]

B012

Arbuscular Mycorrhizal Fungal Diversity in Post-mining Area and Natural Forest Area in Goesan, Korea

Hyeok Park1, Yu-Ra Bae1, Dong-Yeo Kim1, Kang-Hyun Ka2, and Ahn-Heum Eom1*1Department of Biology Education, Korea National University of Education2Division of Wood Chemistry & Microbiology, Korea Forest Research Institute

Arbuscular mycorrhizal fungi (AMF) are one of the most widespread symbionts globally. AMF provide better nutrient absorptivity to host plant, enhance tolerance against plant root pathogen, and also improve resistance against soil contamination by heavy metal. Due to such benefits, AMF are very important in harsh environments such as mines, deserts, or coastal areas. In this study, we compared the difference of AM fungal diversity in post-mining area and natural forest area in Goesan, Chungcheongbuk-do, Korea. We sampled 10 post-mining area soil samples, and 10 natural forest soil samples with host plants. We extracted AMF spores in field soils, and identified spores using morphological characteristics and 18S rDNA sequence analysis. Consequently, we discovered 5 species from 3 genera. Acaulospora was the most abundant genus in post-mining area soils, and Ambispora was the most abundant genus in natural forest soils. These results show that the AM fungal diversity of post-mining area soil is different to diversity of natural forest soil.[Supported by grants from NIFoS]

Poster

www.msk.or.kr | 15

B013

Genome Analysis of Two Strains Belonging to the Genus Limnohabitans, a Major Bacterial Group in Keum River

Joong-hyeon Ahn and Seung Bum Kim*

Department of Microbiology and Molecular Biology, Chungnam National University

From the previous investigation of freshwater microbiome in the four major inland water bodies of Korea using pyrosequencing analysis, the genus Limnohabitans was found as a main bacterial group. In this study, two strains affiliated with Limnohabitans were isolated, and designated 103DPR2 and 63ED37-2. These two strains showed nearly identical relationship with dominant groups of Limnohabitans appearing in different seasons as determined by pyrosequencing. The complete genomes of two strains were analyzed to understand the properties of the strains as a dominant group in the freshwater planktonic bacterial community. Genomic DNA sequences of two strains were analyzed using single molecule realtime sequencing (SMRT) technique, and single, circular contigs were obtained for both strains. The sizes of two genomes were 2.95 Mbp (2,085 CDSs) and 3.37 Mbp (3,180 CDSs), the DNA G+C contents were 53.3 and 59.9 mol%, the number of protein coding genes were 2,085 and 3,180, and the copies of rRNA genes were 6 and 9, respectively. The genome sequences were deposited to GenBank under the accession numbers CP011834 and CP011774. COGs related to amino acid transport and metabolism (category E), and energy production and conversion (category C) appeared as the most dominant metabolic categories for Limnohabitans genomes. This is the first report on the complete genomes of Limnohabitans, which will provide a basis for understanding their dominance in aquatic habitat.

B014

Effects of Urea on Abundance and Community of Nitrite-dependent Anaerobic Methane Oxidation Bacteria in a Rice Paddy

Sang Eun Jeong, Hyo Jung Lee, and Che Ok Jeon*


To investigate the effects of urea on the abundance and community of nitrite-dependent anaerobic methane oxidation (n-damo) bacteria in a rice paddy, different amounts of urea (0, 45, 90, and 180 kg/ha) were applied three times (–1, 15, 45 days of transplantation) to a rice paddy and n-damo abundance and community were investigated using T-RFLP (Terminal-Restriction Fragment Length Polymorphism) after 18 days of third application. Soil cores were obtained and divided into three layers (top, 0–5 cm; middle, 10–15 cm; bottom, 20–25 cm). Abundance of n-damo was not significantly different depending on the amount of urea application. A phylogenetic tree of M. oxyfera-like bacteria based on 16S rRNA genes derived from the rice paddy showed that M. oxyfera-like bacteria were divided into four clades. Using an online program (Restriction Enzyme Picker Online), NciI was selected as a suitable restriction enzyme to discriminate the n-damo clades. The n-damo based on T-RFLP patterns showed that the clades b and c were the most abundant clades in all depths. In particular, the relative abundance of clade c was higher along a depth gradient in all rice paddies. However, the n-damo community was not different depending on the amount of urea application. [This study was supported by grants of the National Research Foundation of Korea (No. 2013R1A2A2A07068946, 2012R1A1B), funded by the Korean Government (MEST).]

B015

Diversity of Bacteria Enriched with Coastal Area Samples Contaminated with Petroleum Oils

Seon-Hee Kim and Hyung-Yeel Kahng*

Department of Environmental Education, Sunchon National University

Coastal area samples contaminated with petroleum oils were collected and used for enrichment culture to isolate bacteria degrading petroleum oils such as gasoline, diesel, and kerosene. During enrichment culture for a couple of months five hundred microliter samples were periodically taken per week and transferred to a sequential enrichment culture in different media containing gasoline, diesel, or kerosene for a couple of weeks. Analysis of bacteria enriched with different petroleum oils showed that bacterial diversity pattern from the diesel medium was similar to that from kerosene medium, but distinguished from that of gasoline medium. Three common representative bacterial genera from both diesel- and kerosene-media were Pseudomonas, Maribacter and Rhodococcus. From the gasoline-media were only a couple of genera, Pseudomonas and Alcanivorax found. Pseudomonas was the only genus obtained from all the media with diesel, kerosene, and gasoline. A total of thirteen genera isolated and identified from the enrichment culture using three kinds of petroleum oils were Alcanovorax, Algoriphagus, Brevundimonas, Demequina, Erythrobacter, Hoeflea, Maribacter, Microbacterium, Phaeobacter, Pseudomonas, Rhodococcus, Shewanella, and Thalassospira.[Suppported by grants from Ministry of Environment]

B016

Isolation and Characterization of Calcium Carbonate Precipitating Bacillus and Sporosarcina Species from Concrete

Hyun Jung Kim and Woojun Park*

Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University

Microbially induced calcium carbonate precipitation (CCP) is a long-standing, but re-emerging environmental engineering process for production of self-healing concrete, bioremediation, and long-term storage of CO2. Three CCP-capable bacteria, Bacillus sp. JH3, Bacillus sp. JH7 and Sporosarcina sp. HYO08 were isolated from two different concrete samples. Morphologically distinct extracellular CCP by the three strains were observed using energy dispersive X-ray spectrometry mapping and field emission scanning electron microscopy. Three strains showed different physiological characteristics: urease activity, exopolysaccharides production, growth at alkali pH, and high salt concentrations. Strain HYO08 had the highest urease activity and was more alkali-tolerant (pH 11). Strain JH7 was more halotolerant (5% NaCl) than two other strains. Barcoded pyrosequencing-based bacterial community analysis of the two concrete samples revealed low abundance (<1%) of Bacillus and Sporosarcina species. High relative abundance of Actinobacteria (<39%) and Cyanobacteria (<12%) indicated possibility of other novel CCP-capable bacteria present in concrete. Strain JH7 having both alkali- and halotolerant ureolytic activity was capable of plugging soil pores and precipitating heavy metals. This study suggests the variance of CCP within different CCP-capable bacteria and the potential of utilizing isolated strains in environmental engineering fields. [Supported by grants from NRF.]


16 | www.msk.or.kr

B017

Genomic and Transcriptomic Analyses of Acinetobacter oleivorans DR1 under Long-chain Alkane

Chulwoo Park, Jaejoon Jung, and Woojun Park*


Genomic analysis revealed the presence of two copies of each alkB-, almA-, and ladA-type alkane hydroxylase (AH) in diesel-degrading Acinetobacter oleivorans DR1. DR1 cells could grow on C12–C30 alkanes, but not on C6–C10 alkanes. Expression analysis of six AHs showed that alkB1 and alkB2 are major AH-encoding genes under C12–C30 alkanes. Unlike other studies, our expression and mutant studies suggested that alkB-type enzymes could be also responsible for lone-chain (LC) alkane degradation. Interestingly, only almA2 was highly inducible among all AH genes under C30 while almA1 was constantly expressed. Expression of two ladA genes was not observed under all tested conditions. RNA-seq analysis under C30 showed up-regulated desaturases, suggesting changing of dynamic membrane fluidity to facilitate adaptation under LC. Highly up-regulated trehalose biosynthesis genes indicate possibility of trehalose lipid as a biosurfactant for LC alkne metabolism. Up-regulation of inorganic ion stress-induced operons such as pho, ssu, and especially kdp genes implies experience inorganic ion-limited conditions probably due to difficulty in accessing charged ions. Phylogenetic and genomic analyses demonstrated that Acinetobacter is rare species possessing multiple alkane systems possibly through horizontal gene transfer. [This work was supported by grants from NRF]

B018

Comparative Genomics Reveals Insights into Adaptation of Ramlibacter Species to Different Soil Habitats

Hyo Jung Lee and Che Ok Jeon*


Members of the genus Ramlibacter have been mainly isolated from various soil habitats. In particular, two Ramlibacter species, R. solisilvae 5-10T and R. tataouinensis TTB310T, were isolated from evidently contrast soil habitats, forest and desert, respectively. Here, the complete genome of strain 5-10 was sequenced and compared with strain TTB310. Strain 5-10 harbors genes related to diverse metabolisms and stable mechanisms, including ribosomal operon, posttranslational modification, protein turnover, chaperones (COG O), and amino acid and lipid transport and metabolism (COGs E and I), and cellular metabolisms, such as sugar, urea, and catechol metabolisms and multidrug resistance genes in high abundances, which may confer good fitness of strain 5-10 to forest soil. Whereas genes related to response mechanisms and exopolysacharride biosynthesis genes, including transcription, signal transduction mechanisms, and carbohydrate transport and metabolism (COGs K, T and G), and tolerance to oxidative stress, such as catalase, peroxidase, thioredoxin and carotenoid biosynthesis, and gliding motility are abundant, which may confer successful adaptation of strain TTB310 to fluctuated hot and dry desert soil environments. This comparative genomic study will provide insights into adaptation of Ramlibacter species to different soil habitats through genomic evolution. [This study was supported by the National Research Foundation of Korea (No. 2013R1A2A2A07068946), funded by MEST.]

B019

Screening of Highly Efficient Nitrogen Fixing Bacteria from Soils and Their Utilization for Plant Growth Promotion

Sun-hwan Jeong and Sang-Seob Lee*

Department of Life Science, Kyonggi University

The utilization of dinitrogen gas (N2) as a source of cell nitrogen is called nitrogen fixation. There are limited number of prokaryotes which can fix N2. The current was focused on the screening of highly efficient free living nitrogen fixing bacteria from soil and their potential to promote the plant growth. A soil sampling survey was conducted to collect the soil samples from Mun-kyeong and Yeon-cheon in South Korea. Initial screening of nitrogen fixing bacteria was conducted through the addition of mannitol and malic acid as carbon sources which help in the enrichment of such bacteria. Essential elements were also added in the growth medium which helps to fix dinitrogen gas (N2). Isolation and purification of diazotrophic bacteria was done by dilution plate technique. Each strain was incubated for 5 days and their nitrogen fixation efficiency was measured by acetylene reduction assay (ARA). In total 102 strains were measured by ARA and three bacteria namely, Cedeacea lapagei GTC 346T, Citrobacter youngae CECT5335T and Enterobacter ludwigii EN-119T were showed to be highly efficient and their nitrogen fixation efficiency was recorded up to 100%, 97.8% and 83.8%, respectively. Further research is focus on the identification of optimal growth conditions such as O2 concentration, temperature, pH and carbon source for high level of nitrogen fixation under filed condition to promote plant growth.

B020

Screening and Isolation of BTEX Degrading Bacteria from Tongyeong Sea Water of Korea

Hye Ji Kim and Sang-Seob Lee*

Department of Life Science, College of Natural Science, Kyonggi University

BTEX refers to the chemicals benzene, toluene, ethylbenzene and xylene. They are the volatile components commonly associate with petroleum products. BTEX can also dissolve in water and it may found in surface and groundwater at contaminated sites. A survey was conducted to collect the sea water and sediments samples from Tong-yeong region of South Korea (Masan gulf waste water treatment, Dot island, Jinhae gulf, and fish raising farm). An enrichment culture was established using seawater to isolate the BTEX degrading bacteria. A total of 85 bacterial strains were screened which have capability to degrade BTEX. Of these 85 strains, 16 bacterial strains were identified which have high degradation efficiency. Out of these 16 strains, a strain designated FDM 14-2 showed 95.78% of benzene degradation, 6.78% of toluene degradation 4.26% of ethylbenzene degradation and 0.62% of p-xylene degradation. Based on 16S rRNA gene sequence analysis strain FDM 14-2 was identified as Bacillus aryabhattai B8W22T.

Poster

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B021

Development of Antifungal Compounds from Streptomyces sp. NF186 against Fusarium oxysporum f. sp. conglutinans

Sung-Jin Cho1 and Sang-Seob Lee2*1Department of Biological engineering, Kyonggi University, 2Department of Life Science, College of Natural Science, Kyonggi University Fusarium oxysporum is a soil borne plant pathogen in the class Hyphomycetes, causes Fusarium wilt. Fusarium wilt caused by Fusarium oxysporum f. sp. conglutinans is a devasting soil-borne disease of cabbage plant in warmer climates that result in a sever reduction in yield.To search the efficient antagonistic bacteria against this pathogen, a soil sampling survey was conducted to collect the samples from agricultural land in South Korea. A dual culture assay was performed to screen the antagonistic bacteria against this pathogen. A total of 1159 bacterial strains were screened for antagonistic activity. Out of these strains only 40 bacterial strains showed formation of clear zone around the pathogen colony .Out of the forty strain only strain designated streptomyces NF186 was selected for further analysis.Small subunit ribosomal RNA (16S rRNA) gene sequencing analysis identified this strain as Streptomyces NF186 highest sequence similarity with Streptomyces lactacystinicus OM 6519T. Optimization of the growth condition for the production of high amount of antifungal compound from strain NF186 is under progress.

B022

Comparative Analysis of Bacterial Communities Isolated from Polychaete Habitat and Non-habitat in a Coastal Wetland Microcosm

Seyeon Shin and Hyung-Yeel Kahng*


Our previous study showed major bacterial species belonging to Bacillus, Vibrio and Shewanella in polychaete, which was known to play an important role as a scavenger in coastal wetland. In order to investigate the impact of ploychaete on the distribution pattern of bacterial communities, a coastal wetland microcosm was constructed, in which polychaete was planted in some compartments of the mesocosm and incubated for a couple of weeks with the periodical addition of sterile feedstuff. Samples which were taken up from the mesocosm were placed and spread on Marine Agar (MA) media to obtain bacterial colonies. Eleven bacterial genera, Bacillus, Bartonella, Gaetbulibacter, Kocuria, Marinobacter, Oceanisphaera, Planomicrobium, Pseudomonas, and Vibrio were found from non-habitat of polychaete, while from polychaete habitat were seventeen genera, Bacillus, Gaetbulibacter, Halobacillus, Idiomarina, Lysobacter, Marinobacter, Marinobacterium, Oceanisphaera, Planomicrobium, Pseudomonas, Rheinheimera, Rhodobacter, Shewanella, Sphingopyxis, Streptomyces, Tenacibaculum and Vibrio found. Chemical analysis of the mesocosm wetland soils showed that soil composition was changing during the life of polychaete planted. These findings suggested that polychaete might make a great influence on change of wetland soil environment as well as increased bacterial diversity in coastal wetland mesocosm. [Supported by Ministry of Food, Agriculture, Forestry and Fisheries]

B023

Gut Microbiota of Mottled Skates, Raja pulchra from the Yellow Sea

Eun Bae Kim

Department of Animal Life Science, College of Animal Life Sciences, Kangwon National University

Raja pulchra, Cham-hong-eo in Korean, is a kind of wild mottled skates. Many Korean people have long enjoyed the unique stimulating flavor from skate ripening. Gut microbiota of the species has never been investigated by using high-throughput sequencing before. Here, we investigated gut microbiota of R. pulchra captured from the Yellow Sea in South Korea. Gut samples were collected from the large intestine of 6 individuals; 3 females (F, 9.1±1.4 kg) and 3 males (M, 5.2±0.4 kg). To analyze microbial profiles, 16S rRNA V4 region from gut contents was amplified by PCR and sequenced using the Illumina MiSeq Sequencer. At the phylum level, relative abundance of Firmicutes (F : M = 51.7% : 40.4%, P=0.096) and Bacteroidetes (F : M = 28.4% : 15.1%, P=0.013) were higher in female skates, but Proteobacteria was more abundant (F : M = 7.9% : 36.7%, P=0.0025) in males. At the genus level, Photobacterium (15.8%), Lactobacillus (13.6%), Prevotella (11.6%) were the most abundant in the skate large intestine. The Photobacterium genus was much more frequent in males (F : M = 2.2% : 29.5%, P=0.011). These results indicate that there are differences between female and male skate gut microbiota. Future research is required to clarify any association between gut microbiota and skate physiology. [Supported by a grant from Marine Biotechnology Program (PJT200620) funded by Ministry of Oceans and Fisheries, South Korea]

B024

Glyoxylate Bypass Governs Bacterial Respiration under Oxidative Stress

Sungeun Ahn and Woojun Park*


The glyoxylate shunt is an alternative to the TCA cycle that bypasses the carbon dioxide-producing steps and is essential for acetate and fatty acid metabolism. The glyoxylate shunt is upregulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, the isocitrate lyase (aceA) and malate synthase (glcB) genes are not in an operon, unlike in Escherichia coli; thus, two genes might exhibit different transcriptional responses to stress. Contrary to our expectations, deletion of aceA from P. aeruginosa improved cell growth under oxidative and antibiotic stress, which could be attributed to decreased NADH production. Transcriptome data suggested that aceA mutants underwent a metabolic shift to aerobic denitrification, supported by additional evidence: upregulation of denitrification- related genes, decreased oxygen consumption without ATP reduction, increased production of denitrification intermediates, NO and N2O, and increased cyanide resistance. A global bioinformatics analysis showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. Our data suggest that the presence of isocitrate lyase, an aerobic adaptation, appears to allow cells to bypass the TCA cycle, not only to preserve the carbon source, but also to tolerate oxidative stress. [Supported by NRF.]


18 | www.msk.or.kr

B025

Endogenous Hydrogen Peroxide Increases Biofilm Formation by Inducing Exopolysaccharide Production in Acinetobacter oleivorans DR1

In-Ae Jang and Woojun Park*

Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological, Engineering, Korea University

In this study, we investigated differentially expressed proteins in Acinetobacter oleivorans cells during planktonic and biofilm growth by using 2-dimensional gel electrophoresis combined with matrix-assisted laser desorption time-of- flight mass spectrometry. We focused on the role of oxidative stress resistance during biofilm formation using mutants defective in alkyl hydroperoxide reductase (AhpC) because its production in aged biofilms was enhanced compared to that in planktonic cells. Results obtained using an ahpC promoter-gfp reporter vector showed that aged biofilms expressed higher ahpC levels than planktonic cells at 48h. However, at 24h, ahpC expression was higher in planktonic cells than in biofilms. Deletion of ahpC led to a severe growth defect in rich media that was not observed in minimal media and promoted early biofilm formation through increased production of exopolysaccharide (EPS) and EPS gene expression. Increased endogenous H2O2 production in the ahpC mutant in rich media enhanced biofilm formation, and this enhancement was not observed in the presence of antioxidants. Exogenous addition of H2O2 promoted biofilm formation in wild type cells, which suggested that biofilm development is linked to defense against H2O2. Collectively, our data showed that EPS production caused by H2O2 stress enhances biofilm formation in A. oleivorans. [Supported by NRF.]

B026

Fungistatic Activity of an α-Aminophosphonate Chitosan Derivative against Aspergillus niger on Controlled Microgravity

Yesupatham Sathishkumar1, Kesavan Devarayan2, Byoung-Suhk Kim3, and Yang Soo Lee4*1Department of Forest Science and Technology, College of Agriculture and Life, 2Department of Basic Sciences, Tamil Nadu Fisheries University, Nagapattinam, India, 3Department of BIN Convergence Technology, Chonbuk National University, 4Department of Forest Science and Technology, College of Agriculture and Life

Human space cruizing have been carrid sevearal decades since Apollo 11 landed on the Moon. Contamination by various living microorganisms within the international space station is ever increasing mainly due to human activity. In order to control of those of contaminants, microorganisms such as fungi and bacteria are important to maintain the well-being of the astronauts during long-term living in aerospace since the immune functions of astronauts are compromised under microgravity. And for the first time control of the growth of an opportunistic pathogen, Aspergillus niger, under microgravity is studied in the presence of α-aminophosphonate chitosan. A low-shear modelled microgravity was used to mimic the conditions similar to space. The results indicated that the α-aminophosphonate chitosan inhibited the fungal growth significantly under microgravity. In addition, the inhibition mechanism of the modified chitosan was studied by UV-Visible spectroscopy and cyclic voltammetry. This work highlighted the role of a bio-based chitosan derivative to act as a disinfectant in space stations to remove fungal contaminants.[This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (1301001076, 1401001209, 1201002578) and supported by the Basic Research Laboratory Program (2014R1A4A1008140) through the Ministry of Science, Ministry of Trade, Industry & Energy (MOTIE).]

B027

A Study on the Biodegradation of Petroleum Oils by Marine Microbial Consortia

Seon-Hee Kim and Hyung-Yeel Kahng*


A total of twenty one bacterial strains were isolated from crude oil- contaminated sand samples collected in Taean Malipo Beach. Three bacterial strains among them, Brevibacterium frigoritolerans strain SHD-34, Pseudomonas brassicacearn subsp. neoaurantiaca strain SHG-4, and Pseudomonas taeanensis strain SHG-7 were selected due to their faster growth rate and used to construct four kinds of microbial consortia, designated KⅡ-1(SHD-34, SHG-4), KⅡ-2(SHD-34, SHG-7), KⅡ-3(SHG-4, SHG-7), and KⅢ(SHD-34, SHG-4, SHG-7). Individual strains and consortia were tested for their growth rate and biodegradation potential in a minimal medium containing each 1% concentration of diesel, kerosene, or gasoline for seven days' incubation, showing that SHG-4 of individual strains grew fastest in diesel, but growth rate of consortium KⅡ-3 was faster than that of SHG-4 in diesel; in kerosene, KⅡ-3 grew fastest among the tested strains or consortia; in gasoline individual strain SHG-7 fastest growth rate. In GC-MS analysis, KⅡ-1 exhibited biodegradation rates approximately 99% of the initial concentration of kerosene; KⅢ, lowest 56.8% biodegradation rate. In gasoline, all individual strains and microbial consortia showed 97-99.8% of the initial concentration. These facts suggested that consortium KⅡ-1 might perform an effective bioremediation of sea water and coastal wetland contaminated with petroleum oils.

B028

Spatial Disturbances in Altered Mucosal and Luminal Gut Viromes of Diet-induced Obese Mice

Min-Soo Kim1 and Jin-Woo Bae2*1Department of Biology, 2Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University

Gut microbial biogeography is a key feature of host-microbe relationships. Here, we conducted a metagenomic study to determine the composition of the mucosal and luminal viromes of the gut and to evaluate the impact of a Western diet on gut viral ecology. We found that mucosal and luminal viral assemblages comprised predominantly temperate phages. The mucosal virome significantly differed from the luminal virome in low-fat diet-fed lean mice, where spatial variation correlated with bacterial microbiota from the mucosa and lumen. The mucosal and luminal viromes of high-fat, high-sucrose “Western” diet-fed obese mice were significantly enriched with temperate phages of the Caudovirales order. Interestingly, this community alteration occurred to a greater extent in the mucosa than lumen, leading to loss of spatial differences; however, these changes recovered after switching to a low-fat diet. Temperate phages enriched in the Western diet-induced obese mice were associated with the Bacilli, Negativicutes and Bacteroidia classes, and temperate phages from the Bacteroidia class particularly encoded stress- and niche-specific functions advantageous to bacterial host adaptation. This study illustrates a biogeographic view of the gut virome and phage-bacterial host connections under the diet-induced microbial dysbiosis. [Supported by grants from the Mid-Career Researcher Program, NRF-2012-Forstering Core Leaders of the Future Basic Science Program]

Poster

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B029

Isolation and Functional Classification of Marine Bacteria in Jeju Coastal Areas Useful for Industrial Purposes

Seyeon Shin1, Dong-Heon Lee2, and Hyung-Yeel Kahng1*1Department of Environmental Education, Sunchon National University2Research Institute for Basic Sciences, Jeju National University

Hundreds of marine microorganisms were isolated from the waters of Jeju coastal areas for the purpose of screening bacteria which can be used for the degradation of seaweeds. Two hundred bacteria were selected based on their enzyme activities detected by the experiment using a variety of substrates. Thirty eight bacterial strains were found to exhibit protease activity; Ten bacterial strains, amylase activity; eleven bacterial strains, cellulase activity; sixty eight bacterial strains, phosphate solubilizing activity; twenty two bacterial strains, auxin formation activity; fifty one bacterial strains, siderophore producing activity. Furthermore, three hundred seventy five bacterial strains were additionally and independently tested for their potential to degrade agar, carrageenan, alginic acid, and chitin which are major components of seaweeds. It was found that one hundred nineteen bacteria strains had the ability to degrade agar, one hundred twenty three bacterial strains, carrageenan, fifteen bacterial strains, alginic acid, and five bacterial strains, chitin. Interestingly, three bacterial strains among them were found to be super-bacteria capable of degradation of agar, carrageenan, alginic acid, and chitin, suggesting that they may be strong candidates used for a variety of industrial purposes in the future. [Supported by Jeju Special Self-Governing Province]

B030

Isolation of Brackish Water Bacterioplankton from Saemangeum by High-Throughput-Culturing Method Based on Cell-sorter Inoculation

Hyoung Tae Cheon, Yeonjung Lim, Suhyun Kim, and Jang-Cheon Cho*


Cultivation of aquatic bacteria is necessary to understand the physiology and ecological importance of diverse bacterial groups. High-Throughput- Culturing (HTC) based on dilution-to-extinction has been successfully applied to isolate representative bacterial groups. In this study, we applied HTC to a brackish water sample collected from the just inside of the Saemangeum reclamation wall. Inoculation of bacteria was performed using a FACS instrument. Bacterial cells were sorted into 9 fractions according to forward and side scatter, and were inoculated directly from the nozzle into low nutrient media, with a final cell density of 3 cells per well. After 7 weeks of incubation at 20°C, microbial growth was detected in approximately 10% of 864 inoculated wells. Phylogenetic analyses of 16S rRNA genes showed that FACS-HTC isolates were distributed in the diverse phyla/classes such as Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, Planctomycetes, Actinobacteria and Verrucomicrobia, and included major bacterial groups representing either marine or freshwater habitats. Particularly, an uncultured subgroup of marine SAR11 clade was isolated. These results showed successful cultivation of previously uncultured marine and freshwater bacterial groups from brackish environment by application of HTC adopting the FACS inoculation. [This work was supported by a grant from the Marine Biotechnology Program (PJT200620) funded by the MOF, Korea.]

B031

Microbial Reduction of Nitrous Oxide under Environmentally Relevant Concentrations

Doyoung Park and Sukhwan Yoon*

Korea Advanced Institute of Science and Technology

N2O has a global warming potential ~300 times that of CO2 and is recognized as the most potent ozone depletion agent. Many different pathways have been identified to contribute to production of N2O; however, the only identified biological sink of N2O is its reduction by Clade I and Clade II N2O reductases. A recent research suggested physiological difference between the organisms harboring two types of N2O reductases, especially in their affinity to N2O. To examine whether Clade I and Clade II organisms actually differ in their capability to consume and survive on trace N2O, the growth of the two representative strains were compared under consistent supply of trace N2O. A chemostat was designed to supply consistent flow of trace (20 ppmv and 200 ppmv) N2O gas as the sole e- acceptor to Pseudomonas stutzeri strain DCP-Ps1, and Dechloromonas aromatica strain RCB, while acetate and NH4+ was continuously supplied with the liquid medium. Unexpectedly, both organisms were able to attain steady-state in the chemostat with the trace N2O (as low as 11.6 ppmv under steady-state) and did not differ significantly in their ability to consume trace N2O. qPCR identified 3.3-fold difference in the cell numbers between the strains when fed 20 ppmv N2O and that strain RCB exhibited 3.6-fold higher per-cell N2O reduction than strain DCP-Ps1. These observations provide unprecedented insight into biological N2O reduction that may be omnipresent in the environment. [Supported by grants from NRF]

B032

A Soil Bacterial Strain Displays Rifampicin Resistance by Inactivation of the Antibiotic

Ho-Jin Jang, Dae-Wi Kim, Do-Hoon Lee, and Chang-Jun Cha*


Rifampicin is an antibiotic used to treat bacterial infections. It is a key component of anti-tuberculosis therapy, stemming from its inhibition of bacterial RNA polymerase. In the present study, we isolated a bacterial strain from soil which was capable of transforming rifampicin. The strain had the 16S rRNA gene sequence similarity of 99.9% with Variovorax guangxiensis. The bacterium designated Variovorax sp. H1 also displayed resistance to rifampicin and was able to grow in R2A media supplemented with rifampicin (100 μg/ml). HPLC analysis showed that the strain completely converted rifampicin to a metabolite within 3 days. Supernatant from 3-day-grown culture broth was also able to transform rifampicin, but resting cells from the same culture did not. The metabolite was further analyzed by LC-MS/MS and tentatively identified to be rifampicin quinone. Antibiotic susceptibility test was performed using a disk containing the metabolite. Comparison of clear zone with that of rifampicin disk indicated that the metabolite lost its antibacterial activity. Our results showed that Variovorax sp. H1 isolated from soil inactivated rifampicin, conferring the antibiotic resistance.


20 | www.msk.or.kr

B033

Specific Fungal Endophyte Resources: Diversity, Characterization, and Comparative Analysis from Contrasting Coastal Environments of Korea

Young-Hyun You1 and Jong-Guk Kim2*1Marine Microorganism Team, National Marine Biodiversity Institute of Korea2School of Life Science, Kyungpook National University

This study analyzed endophytic fungal distribution in three coastal environments of contrasting climatic, geographical, and geological characteristics: the volcanic islands of Dokdo, the East Sea, and the West Sea of Korea. Fungal endophytes were characterized and analyzed with respect to the characteristics of their coastal environments. Fungal endophytes were characterized and analyzed with respect to the characteristics of their coastal environments. For this purpose, common native halophyte communities from the three coastal regions were selected. Furthermore, isolates were characterized by growth in specific salinity or pH gradients, with reference to previous geographical, geological, and climate studies. Unlike East Sea or West Sea isolates, some of the Dokdo Islands isolates showed halophilic traits in high salinity, and many could grow under extreme alkaline conditions. A higher proportion of West Sea coast isolates showed halotolerance compared with the East Sea or Dokdo Islands isolates. These results suggest that unique fungal biota developed because of a close interaction between host halophytes and their specific environment, even within the same halophyte species. Therefore, this study proposes the application of specific fungal resources for the purpose of restoring sand dunes, salt- damaged agricultural land, or industrialization of halophytic plants. [Supported by National Marine Biodiversity Institute Research Program (2016M00400).]

B034

Fish Possess Unique Microbial Communities Shaped by Surrounding Environments and Distinct from Other Vertebrate

Pil Soo Kim1, Jae Bong Lee2, Min-Soo Kim1, Tae Woong Whon1, Dong-Wook Hyun1, Ji-Hyun Yun1, Na-Ri Shin1, Mi-Ja Jung1, and Jin-Woo Bae1*1Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University, 2National Fisheries Research & Development Institute

Gut microbiota contribute to host organism by beneficial effects such as metabolism, immune response as well as ethological aspect. On the one hand, host organisms also select their commensal microbiota that provides advantages to survival or fitness of host. In recent papers, the composition of gut microbiota is shaped by various factors including diets, habitat and phylogeny of the host. So far, the discoveries that suggested with host- microbe interactions and co-evolutionary histories of vertebrate, yet unveiled in the gut microbiota of most of vertebrate species are uncharacterized. In this study, we investigated bacterial communities of distal gut and luminal contents from different habitats among more than 230 fishes, which collect across streams, lakes and sea. Bacterial metagenomic DNA from gut and luminal fluid of each fish species were sequenced by high-throughput next generation sequencing. Network-based analysis and beta diversity plots based on Unifrac distances show salinity of habitats influences bacterial communities of fish. Additionally, fish gut microbiota clustered differently with other vertebrate gut microbiota (mammals, reptiles and birds). Thus, this study using culture-independent technology suggests deep knowledge to understand unrevealed host-gut microbe interaction in fish and surrounding environments, which can provides circumstantial clue for trends of co-evolution in vertebrate and commensal microbes.[Supported by grants from KRF and NFRDI]

B035

A Physiologically, Genetically, and Morphologically Novel Isolated Ammonia-oxidizing Archaeon, Nitrosocosmicus oleophilus, from Terrestrial Sediment

Man-Young Jung1, Heeji Hong1, Eugene L. Madsen2, Md Arafat Islam1, and Sung-Keun Rhee1*1Department of Microbiology, Chungbuk National University2Department of Microbiology, Cornell University, Ithaca, New York, USA

Diverse ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota are thought to be responsible for ammonia oxidation across many terrestrial habitats. In this study, we isolated and characterized an AOA, strain MY3, from coal tar-contaminated forest sediment. Strain MY3 is in clade "Nitrosocosmicus", which is widespread and abundant in terrestrial habitats. The cells of strain MY3 are large “walnut-like” cocci, divide by binary fission along a central cingulum, and have a tendency to form aggregates. Strain MY3 was mesophilic and neutrophilic. An assay of 13C-bicarbonate incorporation into archaeal lipid indicated that strain MY3 is capable of strict autotrophy. The growth rates and yields of strain MY3 increased when the growth medium was supplemented with several saccharides and complex organic compounds. The attachment of cells of strain MY3 to XAD-7 hydrophobic beads and to adsorbent vermiculite demonstrated the potential of strain MY3 to form biofilms, and with this attachment, the nitrification activity increased. The cell surface was confirmed as hydrophobic by the extraction of strain MY3 from an aqueous medium with p-xylene. In this study, to provide fundamental insights into the factors that shape the structure of AOA communities, key ecophysiological properties were established for a novel representative of the Nitrosocosmicus clade. [This research was supported by the Basic Science Research Program through the NRF (2014R1A1A2009901 and 2015R1A4A1041869).]

B036

The Effect of pH on Nitrogen Dissimilation of Shewanella loihica PV-4 and Its Implication in N2O Emission and Nitrogen Retention

Ha Yeon Kim and Suk Hwan Yoon*

Department of Civil and Environmental Engineering, Korea Advanced Institute of Science and Technology

The use of N fertilizers in agricultural soils is the major contributer to emission of N2O, a potent greenhouse gas. Loss of N from agricultural soils due to denitrification is also a serious problem, as it causes an increased demand for N fertilizer and thereby, an increase in flux through the N cycle. Recent studies have suggested that pH may be a crucial factor in regulation of N2O reduction and competition between denitrification (N loss) and respiratory ammonification (N retention). Here, we have used Shewanella loihica strain PV-4 to investigate the simultaneous effects of pH on the ability of the organism as net N2O source or sink and competition between N-loss and N-retention. Strain PV-4 was incubated with 100 μmoles NO3

- and 500 μmoles lactate as e- acceptor and donor, respectively and 44 μmoles N2O was added to 60-mL headspace in 160-ml serum bottles with varying pH. At pH 6.0, transient accumulation of N2O up to 88 μmoles/bottle but no NH4

+ accumulation was observed after NO3

- was completely reduced. At pH 7.0 and 8.0, no transient N2O accumulation was observed and strain PV-4 served as N2O sink and respiratory ammonification activity was upregulated under the high-pH conditions (300 and 1000 μmoles NH4

+ were recovered from reduction of NO3

- at pH 7 and pH 8, respectively). These results suggest that N2O emission reduction and N retention may be able to be simultaneously achieved by controlling pH in the agricultural fields. [Supported by grants from KETEP]

Poster

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B037

Physiological Properties of Bacterioplankton during Phaeocystis Bloom in Polynya of Amundsen Sea, Western Antarctica

So-Jeong Kim1, Jong-Geol Kim1, Joo-Han Gwak1, Hee-Ji Hong1, Woon-Jong Yu1, Md. Arafat Islam1, Soo-Je Park2, and Sung-Keun Rhee1*1Department of Microbiology, Chungbuk National University, 2Department of Biology, Jeju National University

Polynyas, areas of open water surrounded by sea ice, are sites of intense primary production and ecological hotspots in the Antarctic Ocean. We investigated the bacterioplankton response to phytoplankton bloom in Amundsen sea using metagenomic and metatranscriptomic analysis. From metagenomics data, 12 representative bins were obtained based on coverage plotting. At peak bloom, Polaribacter and Oceanospiriilales were dominant (30% and 10%, respectively) with high activity. However, most of bins were 3–6% in the declining phase without predominant bins. Polaribacter contributed mineralization of biopolymer. Most of LMW DOM degradation is mediated by Gammaproteobacteria such as Oceanospirillales (Ant4D3) genomes and SAR92. A comparative metagenomics and metat-ranscriptomic analysis of polynya bacterioplankton associated with different phases of phytoplankton bloom enabled to provide insights into succession of bacteria niches of key bacterioplankton involved in carbon remineralization. [Supported by the Korea Polar Research Institute (KOPRI, PP16020).]

B038

Bioprospecting for Amylase Producing Bacteria from Arctic Sea Samples

Eungyeong Heo1, Haju Park2, Dockyu Kim2, and Eungbin Kim1*1Department of Systems Biology, Yonsei University2Division of Life Sciences, Korea Polar Research Institute

The living organisms have adapted to this extreme conditions to survive. Generally, enzymatic efficiency is proportional to the temperature. However, living organisms at sub-zero temperatures have different suitable temperature for enzyme activities comparing with other organisms. Polar bacteria can serve as an excellent source of cold-active hydrolytic amylases. As an effort to develop new bacterial resources for amylase, we have screened a total of 495 biological samples from Chukchi Sea, for culturable bacteria with cold-active activities of extracellular amylases using MB media containing starch as indicator substrates. The main condition set for selecting the target bacteria was temperature. We selected the one bacterium producing extracelluar amylase at 4°C and uninhabitable at 30°C. The target bacteria identified Pseudoalteromonas spp. by 16S rRNA sequence, named the bacteria Pseudoalteromonassp. strain EH1. To purify the extracellular amylase, the medium was changed to Zobell mediumwith 0.4% starch. After cultivation, the extracellular proteins over 10 kDa size in supernatant were condensed by viva-flow and freeze dried. The condensed proteins were separated by using ionic exchange column (GE Health care, q-column) and size exclusion column with FPLC.[Supported by grants from KOPRI]

B039

Analysis of the Rhizosphere Microbiome of Tomato Cultivars that are Resistant or Susceptible to Bacterial Wilt

Min-Jung Kwak1, Su Yeon Choi2, Ju Yeon Song1, Hyun Gi Kong2, Hyoung Ju Lee2, Seon-Woo Lee2, and Jihyun F. Kim1*1Department of Systems Biology, Yonsei University, 2Department of Applied Biology, Dong-A University

Bacterial wilt is a severe plant disease caused by the soil-borne bacterium Ralstonia solanacearum. Recently, we initiated a whole metagenomic analysis of the rhizosphere communities of two tomato cultivars that are resistant or susceptible to bacterial wilt. Hawaii 7996 is resistant to the disease, while Moneymaker is susceptible. 16S rDNA reads were extracted from the whole metagenome data using blastn against the SILVA database. Taxonomic analysis of the 16S rDNA sequences revealed that the proportion of Flavobacteriia is higher in the rhizosphere of Hawaii7996 than in the rhizosphere of Moneymaker, whereas the proportion of Betaproteobacteria is higher in Moneymaker than in Hawaii7996. Through the scaffold binning we have reconstructed an unknown Flavobacteriaceae genome named as FG1-H7996/R from the whole metagenome data of Hawaii 7996. Results from the comparative genome analysis showed that FG1-H7996/R have more genes associated with inorganic ion metabolism than closely related Flavobacteriaceae species. These differences in the rhizosphere community structure and gene repertoire suggest that specific taxa could influence the plant resistance against the wilt pathogen. [Financial supports from the Next-Generation BioGreen 21 Program (grant no. PJ008201012012) and the Strategic Initiative for Microbiomes in Agriculture and Food (grant no. 914006-04-1-HD020)]

B040

Differential Compositions of Lichen Microbiomes in Cladonia gracilis According to the Positions at Thalli from King George Island, Antarctica

Hyun-Ju Noh1,2, Jang-Cheon Cho2, and Soon Gyu Hong1*1Division of Polar Life Sciences, Korea Polar Research Institute, 2Department of Biological Sciences, Inha University Lichens are symbiotic organisms that are majorly composed of lichenized fungi (mycobiont) and green algae/or cyanobacteria (photobiont). However, lichen thalli also contain highly diverse microbes (microbionts) such as bacteria, archaea, and fungi. Cladonia may provide different micro-environments to the microbial communities of Cladoia gracilis from King George Island, Antarctica. To reveal the microbial communities of different parts of the lichenized fungi, we sectioned thalli from center, intermediate, and marginal positions of colonies to apical, middle and basal parts. Since apical parts are usually more exposed to sun light and wind, and has lower humidity compared to basal parts, we considered this sectioning to represent different conditions. Bacterial 16S rRNA gene and eukaryotic nuclear large subunit (LSU) rRNA gene were analyzed by 454 pyrosequencing method. Apical parts of thalli contained relatively simple bacterial communities, basal parts contained more complex with diverse phyla. LSU sequences were mostly composed of diverse Cladonia genotypes. Algae and cyanobacteria which are related in photosynthesis are represented in different parts of thalli. Although Cyanobacteria OTUs are enriched on basal parts, major algal OTUs concentrated on apical and middle parts. Considering these results, we conclude that different compositions of lichen microbiome according to the vertical position would depend on the micro-environmental conditions.


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B041

Measuring Patterns by Geographical Locations in Marine Metagenome Data Using Newly Adopted Genotyping by Sequencing

Hoon-Je Seong1, Chung-Yeon Hwang2, Hong-Hee Won3, and Woo-Jun Sul1*1Department of Systems Biotechnology, Chung-Ang University2Division of Polar Biology and Ocean Sciences, Korea Polar Research Institute3Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

As handling of the huge quantity of metagenomic data is difficult and time-consuming, we proposed more effective tool to analyze particular sequences related to restriction enzyme sites for targeting reduced numbers of genes rather than to estimate whole genome sequences. In this study, whole genome shotgun sequencing method was compared to adapted GBS procedure for verifying the efficiency. Additionally, single nucleotide polymorphism’s differences were checked by functional categories. GBS–metagenome procedure was demonstrated with marine epipelagic samples from 10 sites during the voyage from East Sea to Bering Sea. These samples were processed by a restriction enzyme “ApeKI” and then sequenced by Illumina HiSeq. In addition to environmental study, just 4 same site samples were sequenced by Illumina MiSeq for whole genome shotgun sequencing to compare GBS method. Microbial pathway abundance patterns were compared between GBS method and general metagenomic method. There were differences of entropy scores in metabolic genes between East Sea and Western Pacific Ocean. Higher entropy values in Western Pacific Ocean represent the genetic differences than those in East Sea abided by environmental alteration and geographical location. GBS-metagenome is very helpful to understand the community resolving budget and time-consuming issues and also identify variations in functional groups through comparing each site.

B042

Comparative Metagenomic Analysis of Microbial Communities in Wheat Nuruk Fermentation

Jeong-Ah Seo1, Minjoo Kim1, Ki Young Yoon2, Min-Jung Kwak2, Jihyun F. Kim2, and Ju Yeon Song2*1School of Systems Biomedical Science, Soongsil University2Department of Systems Biology, Yonsei University

Nuruk is a fermentation starter of Korean traditional turbid rice wines makgeolli and takju. As a natural fermentation starter, Nuruk is made with grain flour and is fermented by various microorganisms such as fungi, yeasts, and bacteria originated from environment. During fermentation, microbial metabolism such as glycolysis could change biochemical conditions of Nuruk. Since microbial composition of Nuruk has a key role in ripening rice wine, the quality of Makgeolli is mainly dependent on Nuruk. Nuruk samples made of different wheat species were collected from each fermentation step. A purification method of metagenomic DNA was established to exclude undesirable wheat genomic DNA and the extracted microbial genomic DNA of Nuruk were subject to metagenomic sequence analysis. Microbial communities were monitored by the targeted amplicon sequencing of the 16s rRNA genes for bacteria and ITS regions for fungi. Dynamics of microbial structures in Nuruk could be characterized by fermentation time and overall microbial communities of samples were decided by initial inoculated microorganisms.

B043

Diversity and Enzyme Activity of Penicillium Species Associated with Macroalgae in Jeju Island

Myung Soo Park1, Seobihn Lee1, Seung-Yoon Oh1, Ga Youn Cho2, and Young Woon Lim1*1School of Biological Sciences, Seoul National University2National Institute of Biological Resources, Environmental Research Complex

Several Penicillium species were isolated in a survey of extracellular enzyme-producing fungi from macroalgae along the coast of Jeju Island of Korea. Penicillium species were identified based on morphological and β- tubulin sequence analyses. In addition, the halo-tolerance and enzyme activity of all strains were evaluated. A total of 28 strains of 19 Penicillium species were isolated. The diversity of Penicillium strains isolated from brown algae was higher than the diversity of strains isolated from green and red algae. The common species were Penicillium antarcticum, P. bialowiezense, P. brevicompactum, P. crustosum, P. oxalicum, P. rubens, P. sumatrense, and P. terrigenum. While many strains showed endoglucanase, β-glucosidase, and protease activity, no alginase activity was detected. There was a positive correlation between halo-tolerance and endoglucanase activity within Penicillium species. Penicillium has the potential to convert macroalgae to useful biomass. Among 19 Penicillium species, three species―P. kongii, P. olsonii, and P. viticola―have not been previously recorded in Korea. [Supported by grants from the Marine BioResource Bank Program of the Ministry of Ocean & Fisheries, Korea.]

B044

Genome Reconstruction for Prediction of Metabolic Potential of Subsurface Archaea in Intertidal Mud Flat SedimentJoo-Han Gwak1, So-Jeong Kim1, Woon-Jong Yu1, Md. Arafat Islam1, Soo-Je Park2, and Sung-Keun Rhee1*1Department of Microbiology, Chungbuk National University2Department of Biology, Jeju National University

Subsurface in various marine and terrestrial anoxic environments harbors diverse archaea which are not cultivated yet. Although the function of those archaea are studied scarcely, heterotrophic lifestyle has been implicated based on organic carbon isotope tracer analysis. Intertidal zone residing between terrestrial and marine habitats provides various niches for microbes. To investigate microbial community structure in mud flat sediment, pyrosequencing of 16S rRNA gene was performed using samples obtained below 2 m of the surface at intertidal zone at the Garolim bay. A total of 27 dominant phyla were identified. Archaea of Miscellaneous Creanarchaeotal group (MCG) was abundant with > 46% while Proteobacteria, Chloroflexi, Actinobacteria and Planctomycetes were in the range of 6-15%. De novo assembly and binning of metagenomic sequences from Illumina were performed for understanding the metabolic potential of subsurface archaea including MCG. Eight dominant archaeal bins were obtained by coverage based binning to reconstruct genomes for assessment of metabolic potential to provide evidence of their niches. This study extends our knowledge on ecology of subsurface archaea.[This research was a part of the project titled "Long-term change of structure and function in marine ecosystems of Korea", funded by the Ministry of Oceans and Fisheries, Korean]

Poster

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B045

Antibiotics Resistant Testing of Vibrio and Oxytetracycline Resistant Bacteria Isolated from Fish Farming Water in Jeju

Son G. Nguyen, Mincheol Kim, Jungman Kim, Nakwon Hwang, and Tatsuya Unno*

Faculty of Biotechnology, College of Applied Life Science, SARI, Jeju National University

Olive flounder (Paralichthys olivaceus) is highly prized, common flatfish in aquaculture in east Asian countries such as Korea and Japan. Several Vibrio species were reported as marine pathogenic bacteria. Due to the intensive use of antibiotics feed additives in aquaculture, Vibrio and other bacteria obtained antibiotics resistance genes and become potential risks to humans. We isolated oxytetracycline resistant bacteria and Vibrio from Olive flounder fish farms in Jeju island (includes sites: In, Discharge, and Sea coastal area near fish farms in triplication), then conducted antibiotic resistant test. The water samples were also tested by specific PCR for detecting some potential pathogens (include Vibrio anguillarum, V. harveyi, and Streptococcus iniae). Total 288 Vibrio isolates were cultured on TCBS agar, and confirmed by genus specific PCR. Antimicrobial susceptibility tests on Mueller Hinton agar indicated that almost Vibrio spp. isolates were sensitive to Chloramphenicol (96.15–100%), some of Vibrio spp. isolates were resistant to Oxytetracycline (6.25–11.54%), Ciprofloxacin (28.21–40.63%), Ceftazidime (0-8.53%), and Ampicillin (13.41–28.21%). On the other hand, significantly more oxytetracycline resistant bacteria were isolated from discharges compared to seawater running in to fish farm (P < 0.05). However, the presence of potential pathogenic bacteria was not observed in these sites.

B046

Anthropogenic Effect on Prevalence of Antibiotic Resistance in Natural Environments

Shalem Raj Padakandla1, Dae-Wi Kim2, Yejin Jang1, Woo Jun Sul2, Chang-Jun Cha2, and Jong-Chan Chae1*1Division of Biotechnology, Chonbuk National University2Department of Systems Biotechnology, Chung-Ang University

Although antibiotic resistant microorganisms are prevalent in natural environments, the causing reason has not been elucidated clearly and human activity is considered as one of important factors. In this study, the prevalence of culturable antibiotic resistant bacteria and resistance-related genes were investigated from upstream to downstream of Han-river with ampicillin, lincomycin, tetracycline, kanamycin, erythromycin, cephalexin, sulfamethoxazole, and ciprofloxin. The population of culturable antibiotic resistant bacteria (ARB) was gradually increased from upstream to downstream. While higher populations of ARB were appeared against ampicillin, licomycin, kanamycin, and cephalexin, relatively lower abundances of ARB were detected against tetracycline, erythromycin, sulfamethoxazole, and ciprofloxin. Antibiotics resistant genes (ARG) and mobile genetic elements (MGE) were also gradually increased in a number and amount from upstream to downstream. Subsequently, the results obtained by culture and non-culture methods indicate that the ARB containing ARG inhabits more prevalently and gene transfer may be caused more frequently downstream of Han-river compared with upstream.

B047

Characterization of Ampicillin Resistant Aeromonas Species Isolated from Domestic Streams

Shalem Raj Padakandla, Yejin Jang, and Jong-Chan Chae*

Division of Biotechnology, Chonbuk National University

The aim of this study was to investigate the diversity of culturable ampicillin resistant bacteria in domestic streams. We collected about 1,600 bacteria from 28 locations in Korea. As a result of identification by 16S rRNA gene sequencing, the genus Aeromonas was predominant occupying 64.3% abundance among which Aeromonas veronii was dominant species. In order to identify the pathogenicity of the strains, PCR was employed to detect aerolysin, serine protease, lipase, cytotoxic enterotoxin, and temperature-sensitive protease. Only 0.17% of the strains contained tested five genes suggesting the low possibility of chronical infection by Aeromonas veronii inhabiting the natural environments. In susceptibility test against various antibiotics by disc diffusion assay, the antibiotic resistance of bacteria gradually strengthened from upstream to downstream of Han-river, representative consecutive sampling location. This supports the anthropogenic effect on antibiotics resistance in natural environments.

B048

Genomic Insight of Bioplastic Production by Sphingobium chungbukense DJ77T

Motakatla Venkateswer Reddy1, Young-Chang Kim2, and Jong-Chan Chae1*1Division of Biotechnology, 2Department of Microbiology, Chungbuk National University

Sphingobium chungbukense DJ77T was isolated as biphenyl degrader and capable of utilizing anthracene and phenanthrene as a sole carbon source. The strain was originally identified as Sphingomonas chungbukensis and reclassified as Sphingobium chungbukense. The complete genome of S. chungbukense DJ77T was determined with Pacbio sequencing platform. The strain has two chromosomes (3.2 Mb and 1.1 Mb) and six plasmids (382 kb, 310 kb, 270 kb, 229 kb, 36 kb, and 19 kb) with 59.5 and 63.9% range of a G + C content. Poly-β-hydroxybutyrate (PHB) was produced inside the cell of S. chungbukens using 4-methoxybenzoic acid as carbon source. Electron microscopy confirmed the generation of granule inside the cell. Moreover, nitrogen limitation promoted PHB production and the physical properties of the produced PHB were analyzed. To better understand the mechanism in genetic level, omics analysis using the genome will be required.


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B049

Microbial Biogeography on the Sedimentary Environment Influenced by the Arctic Paleoclimate

Dukki Han1, Seung-Il Nam2, and Hor-Gil Hur1*1School of Environmental Science and Engineering, Gwangju Institute of Science and Technology, 2Korea Polar Research Institute

The Arctic marine environment consists of various microbial habitats that harbor each local assemblage. The niche preference of microbial assemblages would directly or indirectly reflect the modern environmental change in the Arctic Ocean with oceanographic traits such as sea-ice dynamics, current circulation, and sedimentation. Such ecological niche for the Arctic microbial assemblages may provide putative evidences for geological events in response to the paleoclimate change. The present study describs a microbial biogeography with well-dated high-resolution paleoceanographic records logged by a 5.4 m sediment core in the western Arctic Ocean. Distribution of microbial populations was linked to extraordinary Holocene sedimentary records, suggesting that microbial taxa may be indirectly available to the reconstruction of paleoclimate. Indeed, glycerol dialkyl glycerol tetraethers (GDGTs), the archaeal lipid biomarker known as a specific indicator for Marine Group I Thaumarchaeota (MG-I) has been used for paleoceanography. However, we found the predominance of Marine Group II Euryarchaeota (MG-II) rather than MG-I in the sediment layer (Unit C) containing remarkably higher GDGTs, suggesting that MG-II contributes to the tetraether lipid pool in sediments due to the input of terrigenous matter during the earliest phase of the Mid Holocene. Our results indicate that the interpretation of GDGTs archaeal biomarker needs to be carefully considered for the Arctic paleoceanography.

B050

Identification of a Variant of New Delhi Metallo-β- lactamase, NDM-9-Producing Klebsiella pneumoniae in an Urban River in South Korea

Doris Y. W. Di1, Jeonghwan Jang2, and Hor-Gil Hur1*1School of Environmental Science and Engineering, Gwangju Institute of Science and Technology, 2BioTechnology Institute, University of Minnesota, Saint Paul, MN 55108, USA

Since the first report of the New Delhi metallo-β-lactamase (NDM-1) resistant Klebsiella pneumoniae in 2008, carbapenemase-producing Enterobacteriaceae have been of great concern all over the world due to its multi-resistance encompassing nearly all antibiotics which refer to as ‘super bacteria’. In 2014, a novel variant NDM-9 metallo-β-lactamase was identified from a clinical K. pneumoniae PPH1303, which differed by a single amino acid substitution (E152K) from NDM-1. In our study, three K. pneumoniae (GJ1, GJ2 and GJ3) isolated from Gwangju River were found to possess blaNDM-9 genes. Antimicrobial susceptibility test indicate resistance to aminoglycosides, carbapenems, cephems, folate pathway inhibitors, fosfomycin, and penicillins but susceptible to fluoroquinolones, phenicols, tetracycline and miscellaneous agents. Whole genome sequencing revealed that the IncFII(Y)-like plasmids carry blaNDM-9, aadA2, aph(3’)-Ia, sul1, and dfrA12 genes along with mercury resistance operon are approximately 108-kb. blaLEN-13, blaTEM-1, and blaCTX-M-65, ant(3’)-Ia, fosA, mph(A), aqxA, and oqxB genes were identified. Genes encoding NDM-9 were located between insertion sequences IS15, IS26 and IS15D1 upstream and ISVsa3, IntI1, and resolvase downstream on plasmids. These mobile elements are predicted to be capable of site-specific recombination, transposition and switching of integron cassettes which increase the potential of horizontal gene transfer among microorganisms in the environment.

B051

Genome Sequence Analysis of the Soil Microbe Dokdonella koreensis DS123

HyeonGwon Lee1, Min-Jung Kwak1, and Jihyun F. Kim1,2*1Department of Systems Biology and Division of Life Sciences, Yonsei University2Strategic Initiative for Microbiomes in Agriculture and Food, Yonsei University

Isolated from the soil sampled near Dokdo island, Dokdonella koreensis DS123 is an Gram-negative, motile, non-spore-forming and rod-shaped that belongs to Xanthomonadaceae family. We obtained high quality sequence of 163-depth from PacBio HGAP3. Total bases of the genome amount to 4,446,619 (70.3% G+C), which is predicted to encode 3,533 CDSs, 60 transfer RNAs, and 2 ribosomal RNA operons. We annotated the gene functions by BLAST searches against the Uniref90, GenBank nonredundant, Pfam, COG, and KEGG databases. The 69 core genes are clustered in Xanthomonodaceae family and the genus Arenimonas is the nearest branch in the phylogenetic tree made with the core genes of the family Xanthomonadaceae. Functional categorization of the predicted genes shows that 72.85% of CDSs can be functionally assigned according to the COG category, and 30.77% of CDSs are assigned based on the Subsystem category. Many methylotroph sequences, expected to play roles as the methanol oxidation, and mono-carbon metabolism, were found in DS123. The methanol-oxidation genes oxidate methanol into formaldehyde. In addition to, the genes related with twitching motility, type 6 secretion system, CRISPR are found.

B052

Genome Sequence of Maribacter dokdonensis DSW-8, a Marine Bacterium Isolated from Seawater of Dokdo, an Island of the East Sea in Korea

Jidam Lee1, Min-Jung Kwak1, Soon-Kyeong Kwon1, and Jihyun F. Kim1,2*1Department of Systems Biology and Division of Life Sciences, Yonsei University, 2Strategic Initiative for Microbiomes in Agriculture and Food, Yonsei University

Maribacter dokdonensis DSW-8T, isolated from the seawater of Dokdo in the East Sea, Korea, is an aerobic rod-shaped Gram-negative bacteria belonging to Flavobacteriaceae. To analyze the genomic features of Flavobacteriaceae bacterium isolated from seawater near the island, we have determined the genome sequence of DSW-8 using Illumina sequencing platform. After read trimming, we have obtained 18,494,287 high-quality reads and with 416-fold coverage of the genome size. After de novo assembly and gene prediction, 16 contigs with 4,434,543 bp (35.95% G+C content) were generated and 3,835 protein-coding sequences, 36 transfer RNAs, and 6 ribosomal RNAs were detected. A phylogenetic tree based on the 82 core genes among 181 sequenced members of the family Flavobacteriaceae showed that DSW-8 forms a sister clade with Maribacter forsetii DSM 18668. In the genome of DSW-8, the genes encoding the proteins associated with gliding motility, molybdenum cofactor biosynthesis, and several kinds of carbohydrate utilization. Moreover, the genes encoding TRAP-type C4-dicarboxylate transport system were detected as DSW-8 specific genes. Gene content of DSW-8 may provide the useful information in the carbon utilization of the marine flavobacteria.

Poster

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B053

Stimulation of Biomass and Lipid Productivity of Marine Diatom Chaetoceros Strains by Utilizing Mud and Food Waste Mixture

Si Wouk Kim1,2*, Moon Jong Kim1, and Geun Ho Gim2

1Department of Energy Convergence, Chosun University 2Department of Environmental Engineering, Chosun University

High biomass and lipid productivities of marine microalgal strains were obtained through mixotrophic cultivation [1]. The cost effective mixotrophic medium for mass cultivation of Chaetoceros sp. KMMCC1319, Chaetoceros fragilis KMMCC1122, and Chaetoceros gracilis KMMCC674, which were used as a food for shell-fish, were prepared by mixing mud clay and food waste in the distilled water. Comparing with the biomass and lipid productivities of Chaetoceros strains cultivated in a f/2 medium (used as a control), those were increased about two-fold in a mud-distilled water mixture. The biomass productivity increased three-fold when the cells were cultivated in a food waste-distilled water mixture, whereas lipid productivity decreased significantly. Interestingly, the biomass and lipid productivities of Chaetoceros strains increased 10 times when they were cultivated in a mud-food waste-distilled water mixture (although the mixture ratios are different depending on microalgal strains). In addition, from the analytical data of mud clay or food waste, it was found that mud contains high concentrations of microelements, humic and fulvic acids, whereas food waste contains high concentrations of nitrogen, phosphorous and organic compounds. Hence we conclude that, these components can stimulate the biomass and lipid productivities of marine microalgal strains when grown under mixotrophic condition.

Reference[1] G. H. Gim, J. Ryu, P. I. Kim and S. W. Kim, Effects of carbon source and

light intensity on the growth and total lipid production of three microalgae under different culture conditions, J. Ind. Microbiol. Biotechnol. DOI: 10.1007/s10295-016-1741-y.


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C001

3D Structure of Family 5 Extracellular Solute-binding Protein in Bifidobacterium longum KACC 91563

Junsang Ham, Han-ha Chai, Hyoun Wook Kim, Bu Min Kim, and Mi-Hwa Oh*

National Institute of Animal Science, RDA

We found Bifidobacterium longum subsp. longum KACC 91563 alleviates food allergy in animal model, and examined ESBP (extracellular solute binding protein) in Bifidobacterium longum subsp. longum KACC 91563 alleviates food allergy through mast cell suppression in a previous research. We tried to understand 3D structure of the ESBP. Protein family, domain, and biological process of ESBP in Bifidobacterium longum subsp. longum KACC 91563 were compared to that of Bifidobacterium longum subsp. longum JDM 301, but partial similarity was found. 3D structure of ESBP in Bifidobacterium longum subsp. longum KACC 91563 was predicted on the basis of the ESBP in Salmonella enterica subsp. enterica serovar Typhimurium which has 40.4% similarity. ESBP in Bifidobacterium longum subsp. longum KACC 91563 may bind to a ligand peptide (Lys-Leu-Lys) weaker than that of Salmonella enterica subsp. enterica serovar Typhimurium. Allergy alleviation could be a strain-specific characteristic of beneficial microbes.

C002

Genetic Strategies for Pikromycin Over-production in Streptomyces venezuelae

Joon-Sun Choi, Ji-Eun Kim, and Jung-Hye Roe*

School of Biological Sciences, College of Natural Science, Seoul National University

Pikromycin is the 14-ring macrolide antibiotic, produced by type 1 PKS system in Streptomyces venezuelae ATCC15439. It is known as a potential raw material for ketolide compounds. For pikromycin over-production, we established host engineering strategies which include promoter change and removal of unnecessary genes. As the first step, we examined the time course of pikromycin production in submerged culture condition. Pikromycin began to appear from the beginning of the stationary phase and accumulated throughout the whole culture period. For over-expression of the pikromycin biosynthetic gene cluster during the stationary phase, we selected several promoter candidates exhibiting strong activities after late exponential phase. We identified transcriptional activities of promoter candidates by using GUS (β-glucuronidase) reporter system and S1 nuclease mapping assay. Through transcriptome analysis of S. venezuelae ATCC15439, we also selected highly expressed gene clusters for putative secondary metabolites. Using homologous recombination, we deleted three gene clusters predicted to encode polyketide biosynthetic enzymes. This triple mutant produced higher level of pikromycin than the wild-type.

C003

Secondary Metabolites for Plant Growth Promoting Produced by Rhodobacter capsulatus PS-2

Ki Moon Bong1, Jong Min Kim1, In Cheol Park2, Chul Won Lee3, and Pyoung Il Kim1*1Jeonnam Bioindustry Foundation, Bio Control Research Center2Agriculture Microbiology Division, National Academy of Agricultural Science3Department of Chemistry, Chonnam National University

Hormones of plant growth promoting (PGP), which are produced in small quantity, affect various activities in plant growth and development. PGP was the effect on crops productivity in fields of agricultural industry. There are several types of auxin (indole-3-acetic acid; IAA), gibberellin (GA3) and abscisic acid (ABA). In this study, Rhodobacter capsulatus PS-2 strain was isolated from paddy soil. The growth rate and PGP production of R. capsulatus PS-2 were investigated under different culture conditions. To measure the productivity of PGPs, LC-MS/MS method was performed in the multiple reaction monitoring (MRM) modes. MRM was employed for qualitative measurement. The MRM transitions were monitored as 176→130 for IAA, 265→148 for ABA and 347→238 GA3, respectively. The results showed that the R. capsulatus PS-2 produced PGP, IAA, GA3 and ABA for plant growth promoting. [Supported by grant from RDA.]

C004

Cultivation Conditions for Mass Production of an Antifungal Lipopeptide from Bacillus methylotrophicus GH1-13

Jong Min Kim1, Ki Moon Bong1, Gong Min Kim1, JaeKyeong Song2, Chul Won Lee3, and Pyoung Il Kim1*1Jeonnam Bioindustry Foundation, Bio Control Research Center2Agriculture Microbiology Division, National Academy of Agricultural Science3Department of Chemistry, Chonnam National University

In this study, optimal culture condition for the mass production of antifungal lipopeptide producing microbe was achieved. Microorganism was kindly provided by Korean Agriculture Culture Collection (KACC), Republic of Korea. The optimal culture conditions such as pH, inoculum volume and medium composition were established. Medium composition was selected by the concentration gradients of yeast extract, glucose, NaCl, K2HPO4, Na2CO3, MgSO4. After mass cultivation, cell-free supernatant was used to assay antifungal activity using the paper disc method and the antifungal lipopeptides were identified by LC-MS/MS (SCIEX API 2000 series). Antifungal lipopeptides of molecular weight 1044.9 Da, 1486.3 Da and 1506 Da were analyzed. From the generated protonated ion peaks in LC-MS/MS, 1044.9 m/z and 1486.3 m/z, 1506.0 m/z were identified as surfactin and fengycin, respectively.[Supported by grants from RDA]

Poster

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C005

Multi-drug Resistant Staphylococcus aureus Growth Inhibition by Violacein Produced by Pseudoduganella Natural Isolated Strain NI28

SooYeon Kim, SeongYeol Chio, and Robert J. Mitchell*


Microorganisms are causing all kinds of human diseases varying from infections to cancer. Nowadays the overuse of antibiotics resulting in multi-drug resistant organism is becoming critical threats to health issues, however, few solutions are found to fix this problem. For the development of therapeutic material, therefore, we should consider both to cure diseases by antibiotic development or rediscovery of traditional one as well as not to build drug resistance. In this study, we introduce the multi-use agent, violacein with variety of profitable effects such as anticancer, antibiotic, antifungal and antiprotozoan effect. The violacein is produced by bacterial strains, particularly Pseudoduganella sp. NI28 strain for this study. Pseudoduganella sp. NI28 strain, violacein producer, is found in soil and has 98% 16S rRNA similarity to Pseudoduganella violaceinigra YIM31327. The violacein has antibacterial activity against Staphylococcus aureus ATCC25923 and other multi drug resistant S.aureus isolated from hospital patient. Using ethanol extraction, violacein is purified. Antibacterial effect of purified violacein was tested on Staphylococcus strains. As a result, minimal inhibitory concentrations of violacein were 5 mg/L in Mueller Hinton broth and 0.62 mg/L in M9 modified media. Hence, antibiotic effect of violacein against multi-drug resistant Staphylococcus aureus has shown possible development of treatment for the multi-drug resistant gram positive bacterial infection.

C006

Impact of Silicate Solubilizing Burkholderia ebumea CS4-2 on Rice Growth

Sang-Mo Kang, Raheem Shahzad, Ko-Eun Lee, Yeon-Gyeong Park, Ah-Yeong Kim, Chang-Woo Seo, Sajjad Asaf, and In-Jung Lee*

School of Applied Biosciences, Kyungpook National University

The current study was aimed to isolate and identify the plant root-associated rhizobacteria and to investigate their potential for silicate solubization, growth promotion, IAA Production and encouragement of silicon uptake and deposition inside the plant. Bacterial isolates were selected on the basis of their silica solubilizing potential and were identified on micromorphological observation and biochemical characterization. Selected bacterial isolate was submitted to NCBI and was identified as Burkholderia ebumea CS4-2. GC-MS results revealed the ability of Burkholderia ebumea CS4-2 to produce high amount of IAA at pH 8. Plant microbe experiment showed a significant positive correlation between Burkholderia ebumea CS4-2 and silica. Bacterial inoculation with the combination of silica significantly promoted all growth attributes as compared to the water treated plants and silica fertilized plants. Microscopic observations demonstrated a significant difference in silicon on leaf epidermis, indicating that Burkholderia ebumea CS4-2 with correlation of silicon resulted in higher Si deposition then silicon fertilized and water treated plants. The current study concluded that the Burkholderia ebumea CS4-2 has the ability to produce IAA, solubilize silicate and promote plant growth. This can be the best alternative of synthetic fertilizer as well as in environment clean up.[Supported by grants from NRF]

C007

Growth Inhibition of Multidrug Resistant Staphylococcus aureus Using Cotton Fabric with Violacein Derivative

SeongYeol Choi1, HeeUn Kwon1, SooYeon Kim1, YeongMi Kwon2, ChangSeok Lee2, JinHyeng Lee3, and Robert J. Mitchell1*1School of Life Science, Ulsan National Institute of Science and Technology2YeeJoo Research Institute, YeeJoo Corporation3Korea Institute of Ceramic Engineering and Technology

Violacein is one of antibacterial pigment produced by various natural bacteria such as J. lividum, C. violaceum. It Inhibits gram positive bacterial growth and kills protozoans which consume bacteria in nature. Even this very powerful advantage, however, violacein and its derivatives are too hydrophobic to dissolve aqueous phase. This study focus on its antibacterial application. The violacein can block gram positive bacteria moreover it can block growth of methicillin resistant Staphylococcus aureus (MRSA) and multi-drug resistant Staphylococcus aureus (MDRSA) using MIC 5 mg/L and one of violacein derivative called deoxyviolacein can effect in dyed cotton. The violacein dyed cotton is shown very low antimicrobial effects (less than 20%) but deoxyviolacein dyed cotton has 99.9% of MRSA growth inhibition following KSK0693 methods. Thus deoxyviolacein, violacein derivative can be used for antibacterial fabrics.

C008

Isolation of Lipase Genes from Goat Ruminal Metagenomic Libraries

Mi-Ra Kwon1, Keun-Sung Kim2, Jin-Sung Lee3, Mi-Rim Park1, Haesu Ko1, and Kyung-Tai Lee1*1Animal Genomics and Bioinformatics Division, National Institute of Animal Science, Rural Development Administration

2Department of Food Science and Technology, Chung-Ang University3Department of Biological Sciences, Kyonggi University

Feed additive is an important pricing decision factor for the animal products in the Korean livestock industry. Most feed enzymes, such as phytase, lipase, protease, carbohydrase, depend on import in Korea. Ruminal microbiome is majorly responsible for digestion in ruminants. In particular, ruminal microbiome in goat has been evolved for digestion of wood roughage. To screen and isolate more efficient feed enzymes, metagenomics fosmid libraries were constructed from ruminal microbiome of three goats. In this study, we focused on the lipase genes as a kind of feed enzymes. A total of 57,600 fosmid clones were used for screening lipase activity. These clones were inoculated on the LB agar plates containing antibiotics of 25 μg/ml chloramphenicol and enzymatic substrate of 1% tributyrin using VIAFLO 384 and grown at 37°C for 48 h. Circular clear zone around clone indicated lipid lysis activity. A total of 188 lipase active clones were screened and 39 clones among them presented strong activity. Strong lipase active fosmid clones were sequenced by shotgun pyrosequencing using GS Junior. Sequence reads were assembled using de novo assembler in Newbler package and MetaGeneMark web program was used for gene annotation. Finally lipase genes were isolated by PFAM and BLASTP search. These lipase genes should be developed for industrial application such as cosmetics, pulping, lubricants as well as feed additive.


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C009

Functional Analysis Of Intracellular Nitric Oxide during Development in a Filamentous Fungus

Anchalee Pengkit1, Sung-Sil Jeon2, Soo Ji Son3, Jae Ho Shin3, and Gyungsoon Park1,2*1Plasma Bioscience Research Center, Kwangwoon University2Department of Electrical and Biological Physics, Kwangwoon University3Department of Chemistry, Kwangwoon University

Studies on nitric oxide (NO) are very rare in fungi compared to mammals and plants. In this study, we investigated production of intracellular NO and its possible roles during development of Neurospora crassa, a model filamentous fungus. Intracellular NO was detected in hypha 8–16 hours after incubation in Vogel’s minimal liquid media and conidiophores during conidiation using a fluorescent indicator (DAF-FM diacetate). NO scavenging inhibited the branching of hyphae and delayed conidiation. The exogeneously added nitric oxide enhanced hyphal extension on VM agar media and conidia formation. NO scavenging reduced transcription of con-10 and con-13, genes preferentially expressed during conidiation. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP), No. NRF-2013R1-A1A3011245 and No. 2010-0027963.]

C010

Identification of Protease Genes from Goat Ruminal Metagenomic Libraries

Mi-Ra Kwon1, Kyung-Tai Lee1, Keun-Sung Kim2, Jin-Sung Lee3, Mi-Rim Park1, and Haesu Ko1*1Animal Genomics and Bioinformatics Division, National Institute of Animal Science, Rural Development Administration, 2Department of Food Science and Technology, Chung-Ang University, 3Department of Biological Sciences, Kyonggi University

Proteolytic enzymes or proteases are degradative enzymes, catalyzing the hydrolysis of proteins. Protease is the most important industrial enzyme, accounting for approximately 60% of the total enzyme market in the world because proteases are widely used in food industries, leather, meat processing, cheese making, digestive, medical treatment, etc. Also, protease is one of major feed enzymes in the livestock industry. In this study, we screened and identified several proteases from metagenomics fosmid libraries of goat microbiomes that have been evolved for digestion of wood roughage. A total of 51,840 fosmid clones were used for screening protease activity. Fosmid clones were inoculated on the LB agar plates containing antibiotics of 25 μg/ml chloramphenicol and enzymatic substrate of 1% skimmilk using VIAFLO 384 and grown at 37°C for 48 h. Circular clear zone around clone indicated skimmilk lysis activity. A total of 29 protease active clones were screened and 9 clones among them presented strong activity. Strong protease active fosmid clones were sequenced by shotgun pyrosequencing using GS Junior. Sequence reads were assembled using de novo assembler in Newbler package and MetaGeneMark web program was used for gene annotation. Finally lipase genes were isolated by PFAM and BLASTP search. These protease genes should be developed for industrial application.

C011

Effect of Structure and Molecular Weight on the Permeabilizing Ability of Polyethyleneimine (PEI)

Soh M. Sandrine and Robert J. Mitchell*


Polyethyleneimine (PEI) is used in a wide range of formulations ranging from washing agents to microbicidal compositions. It is a weakly basic aliphatic polymer known to permeabilize bacterial membranes, property which makes it useful for a wide range of applications. Some studies reported the existence of a difference in degree of permeabilization of bacterial membrane when using PEI of different molecular weight and structures. We hereby in this study try to evaluate the difference in relative fold induction possibly observed between PEI of various molecular weights and structures.

C012

Characterization of Lactobacillus salivarius Strain Isolated from Piglet Feces for Probiotic Uses

Gwi-Deuk Jin1, Jongbin Park2, and Eun Bae Kim1*1Department of Animal Life Sciences, Kangwon National University, 2Department of Animal Life System, College of Animal Life Sciences, Kangwon National University

Lactobacillus salivarius is one of lactic acid bacteria used as a beneficial probiotics for human or animal gut. Some L. salivarius strains can inhibit growth of pathogenic bacteria when they live in the human/animal guts. In this study, we isolated L. salivarius strains from piglets feces in South Korea and characterized their probiotic potentials. We isolated 500 lactic acid bacteria colonies from feces of 10 post-weaning piglets using MRS agar plates. Among them, we identified 120 Lactabcillus strains including L. salivarius, Lactobacillus plantarum and Lactobacillus reuteri by using Multiplex-PCR, and 50 L. salivarius strains were selected for further analyses. Out of the 50 strains, we selected 1 strain, called LS.EBK here, have a high antimicrobial activity against pathogenic Salmonella enterica serovar Typhimurium. LS.EBK strain showed similar growth with other L. salivarius strains. Survival rates of the strain exposed to MRS broth HCI-adjusted to pH 3.0 (for 30 min) and MRS broth containing 0.5% of bile salt (for 30 min) were 0.77% and 0.68%, respectively. These results indicate that L. salivarius LS.EBK strain have a potential characteristics for probiotic uses in livestock industries. In the future, we will verify its probiotic effects on piglets by animal trials.[This study was supported by the Strategic Initiative for Microbiomes in Agriculture and Food, Republic of Korea (914005-04).]

Keywords: Probiotic, Lactobacillus salivarius, Salmonella, Antimicrobial

Poster

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C013

Single-stranded DNA Aptamers Targeting Antimicrobial Peptides: PG1, PR26 and PMAP36

Phat-Loc Nguyen1, Kyeoung-Ah Lee1, Simranjeet Singh Sekhon1, Jiho Min2, and Yang-Hoon Kim1*1Department of Microbiology, College of Natural Sciences, 2Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University

Aptamers are capable of binding to large molecules such as peptides using in detection or chemical modification. In this study, aptamers with 100 bases long variable region are employed to target 3 peptides: PG1 (Protegrin-1, an antimicrobial agent in the treatment of local or systemic infections); PR26 (Proline-arginine-rich Neutrophil Antibacterial peptide, against enteric gram-negative bacteria) and PMAP36 (Porcine Myeloid Antibacterial peptide, a highly cationic and amphipathic α-helical peptide) which are identified as important components of host defense mechanism [1-3]. After 10 rounds of selection, ssDNA aptamers with high affinity were generated by SELEX (Systematic Evolution of Ligands by Exponential enrichment) and were analyzed using mfold-DNA folding form and surface plasmon resonance (SPR) assay. [This research was supported by Bio-industry Technology Development Program for, Ministry for food, Agriculture, Forestry and Fisheries, Republic of Korea (311007-5).]

C014

Selection and Characterization of the Glypican-3 Binding DNA Aptamers

Quang-Thai Nguyen1, Kyeong-Ah Lee1, Sinranjeet Singh Sekhon1, Yang-Hoon Kim1, Sung-Jin Cho2, and Jiho Min3*1Department of Microbiology, 2Department of Biology, 3Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University

Glypicans are heparan sulfate proteoglycans linked to the cell surface and play an important role in regulation of cell growth, differentiation, and migration. While six members of the glypican family, Glypican-3 is an efficient diagnostic marker of human hepatocellular carcinomas. Aptamers are single-strand oligonucleotides able to bind specifically and sensibility to target molecules, offering great potential for applications in diagnosis and therapy. In addition, aptamers generally have higher specificity and stability for their targets than corresponding antibodies. ssDNA aptamers were selected using the Systematic Evolution of Ligands by EXponential enrichment methodology from an initial library containing a 40 bases long variable region. After in vitro selection, the structural stability as affinity ligands of these aptamers specifically were quantified by strict criteria of equilibrium (Kd) using surface plasmon resonance and compared with the corresponding monoclonal antibody.[This study was carried out with the support of "Cooperative Research Program for Agricultural Science & Technology Development (PJ011661)", Rural Development Administration, Republic of Korea.]

C015

Deoxyviolacein Mass Production and Aggregation Using Fermenter with Escherichia coli Culture

HeeUn Kwon, SeongYeol Choi, and Robert James Mitchell*

School of Biological Science, Ulsan National Institute of Science and Technology

Violacein is well known antibiotic dye which have effect on antimicrobial, anti-cancer, antiprotozoan and antiviral areas. Deoxyviolacein is one of violacein derivatives which is produced by various natural violacein producing strain such as J.lividum, C.violaceum, Duganella sp., Pseudoduganella sp., and Collimonas sp. It have been discussed that the mass production of violacein and its derivative have important value to study. This study is focused on the simple mass production of deoxyviolacein with high density cell culture of E. coli and the aggregation of deoxyviolacein in cell culture, making it harder to measure exact deoxyviolacein concertration while culturing. We made W3110 BH2 strain containing deoxyviolacein-producing and bacterial hemoglobin plasmid. This strain presents 0.86 g/L of deoxyviolacein production for 20 hours culture in broth. Total deoxyviolacein amount produced including aggregates is 1.12 g/L in 24 hours.

C016

Adaptive Laboratory Evolution of Leuconostoc mesenteroides J18 in Response to Low Temperature

Hye Rim Kim1, Ga Yeon Jo2, Hey Hee Jeon2, and Che Ok Jeon2*1Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, and Center for Food and Bioconvergence, Seoul National University2Department of Life Science, Chung-Ang University

Lactic acid bacteria (LAB) such as Leuconostoc mesenteroides and Leu. citreum have been used as kimchi starters to produce kimchi with uniform quality and good organoleptic properties. However, the use of kimchi starters does not guarantee successful kimchi fermentation because of the competition failure of kimchi starters with indigenous LAB under fermentation conditions. Because kimchi is generally fermented at low temperatures (0–8°C), an adaptive laboratory evolution approach was used to improve the low temperature adaption. The subcultures of Leu. mesenteroides J18 in CDM medium were performed 69 and45 times representing 300 and 200 generations (total 500 generations) at 8 and 6°C, respectively. To characterize the low temperature adaption of strain J18, three substrains with subcultures of 50, 200, and 500 generations were selected and their growths at 6°C were compared with wild type. The genomes of three substrains were sequenced and compared with that of wild type to identify genes conferring the low temperature adaption. As the results, some unique nucleotide changes were identified from the genomes of three substrains, including genes encoding 4-aminobutyrate aminotransferase and exopolysaccharide biosynthesis. In addition, we will discuss the genetic insights into the low temperature adaption of strain J18 in the poster section. [This study is supported by the Strategic Initiative for Microbiomes in Agriculture and Food, Ministry.]


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C017

Activation of Endophytic-plant Growth-promoting Bacteria (EPGPB) by Dielectric Barrier Discharge Plasma Treatment

Sang hye Ji1, Se Chul Chun2, Eun Ha Choi1, and Gyungsoon Park1*1Plasma Bioscience Research Center, Kwangwoon University, 2Department of Bioresources and food science, College of Life and Environmental Sciences, Konkuk University

We investigated the potential of micro Dielectric Barrier Discharge (DBD) plasma to increase the effect of isolated endophytic-plant growth-promoting bacteria (EPGPB) on rice plants. To find the best conditions for the activation of EPGPB by plasma treatment, bacteria activity was investigated in accordance with the feeding gases, plasma treatment time and incubation time. The activity of EPGPB was enhanced more by Air than N2 plasma treatment and also we were able to confirm the rice seed germination rate and plant growth-promoting activity by Air plasma treated EPGPB. The levels of ROS and RNS from plasma and energy were different depending on the kinds of gas supplied to DBD plasma and these might work as critical factors in activating the EPGPB by the plasma. The colonization of Air plasma-treated EPGPB were increased within rice plant compare to untreated control. Through the SEM analysis, the attachment ability of Air plasma-treated EPGPB on rice seed surface was confirmed higher than untreated control. We will study on the mechanism for resistance to fungal pathogen about the plasma-treated bacteria from now on. Induced disease resistance ability by plasma treatment in EPGPB is a very important factor as a development of biocontrol agent.

C018

Inhibition Effect of Lactobacillus plantarum in Colon Cancer

Anna Jeong, Sooyeon Song, and Sejong Oh*

Division of Animal Science, Chonnam National University

Colorectal cancer is the leading cause of cancer death in Korea because of the introduction of Western dietary habits. Particular Lactobacillus strains have beneficial bioactivities in the gastrointestinal tract. The production of intracellular reactive oxygen species (ROS) and the amounts of intracellular calcium, protein kinase C activity, cytochrome c, Bid, Bcl-2, Bax, and the apoptosis-mediated proteins (caspase-8, caspase-3, and poly ADP ribose polymerase [PARP]) were evaluated to understand the induction of programmed cell death in HT-29 cells by L. plantarum. The results obtained from this study indicated that the relative intensities of the apoptotic-related factors (intracellular ROS and intracellular calcium) and of apoptotic signals (Bax and t-Bid) increased with increasing concentrations of the membrane proteins isolated from heat-killed L. plantarum, whereas the relative intensities of cytochrome c, Bcl-2, caspase-8, caspase-3, and PARP decreased. This study determines whether proteins (12 kDa and 15 kDa) isolated from heat-killed Lactobacillus plantarum induce programmed cell death in HT-29 cells. Proteins isolated from L. plantarum can stimulate the apoptotic signals and then consequently induce programmed cell death in HT-29 cells. The results in this study suggest that the proteins isolated from L. plantarum could be used as an anti-tumoral agent in probiotics and as a component of supplements or health foods.

C019

Anti-tumor Effect of L-asparaginase Delivered by Salmonella typhimurium on Solid Tumors

Kwangsoo Kim, Daejin Lim, Yeongjin Hong, Kun-Hee Kim, Kyeong il Park, Shinnam Li, Hyun-Ju Kim, Hyon E. Choy, and Jae Ho Jeong*

Department of Microbiology, Chonnam National University Medical School

Bacteria can be engineered to deliver anticancer proteins to tumors via a controlled expression system that maximizes the concentration of the therapeutic agent in the tumor. L-asparaginase (L-ASNase), which converts asparagine to aspartate and glutamine to glutamate, is an anticancer protein used to treat acute lymphoblastic leukemia. In this study, Salmonellae were engineered to express L-ASNase selectively within tumor tissues using the inducible araBAD promoter system of E. coli. Antitumor efficacy of the engineered bacteria was demonstrated in vivo in solid malignancies. This result demonstrates the merit of bacteria as cancer drug delivery vehicles to administer cancer-starving proteins such as L-ASNase to be effective selectively within the microenvironment of cancer tissue.

C020

Proteobacteria: Diagnostic Marker for Dysbiosis in Gut Microbiota

Na-Ri Shin, Tae Woong Whon, and Jin-Woo Bae*


Recent advances in sequencing techniques, applied to the study of microbial communities, have provided compelling evidence that the mammalian intestinal tract harbors a complex microbial community whose composition is a critical determinant of host health in the context of metabolism and inflammation. Because an imbalanced gut microbiota often arises from a sustained increase in abundance of the phylum Proteobacteria, the natural human gut flora normally contains only a minor proportion of this phylum. Here, we review studies that explored the association between an abnormal expansion of Proteobacteria and a compromised ability to maintain a balanced gut microbial community. We also propose that an increased prevalence of Proteobacteria is a potential diagnostic signature of dysbiosis and risk of disease. [This work is supported by a grant from the Mid-career Researcher Program (2011-0028854) through the National Research Foundation of Korea (NRF) and a grant from the Strategic Initiative for Microbiomes in Agriculture and Food, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.]

Poster

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C021

Screening and Fermentation Characteristics of Bacillus sp. with High Amylase and Protease Activity Isolated from Traditional Nuruks

Min Ju Park, Sung Wook Han, Su Jin Heo, Young Ho Hong, Seong Jun Cho, and Seung Won Park*

Life Ingredient & Material Research Institute, CJ Cheil Jedang

Enzyme producing microorganisms were isolated from many kinds of Korean traditional foods. Among the total 1,500 isolated strains, a strain isolated from Nuruks, designated as BA245 was determined to have the most potent ability to present high enzymatic activities for α-amylase and protease. Enzyme activity was examined by using 1% soluble starch and 2% skim milk agar plates. 16S rRNA and gyrase A gene sequence analysis revealed that strain BA245 belongs to the Bacillus amyloliquefaciens. The optimal activities of its α-amylase and protease were observed at pH 7.0–9.0 and 37°C. After solid-state fermentation of BA245 on mixed grain for 24 h, the α-amylase and protease activities were 2,762 and 3,868 Units/g. The contents of less than 30 kDa peptides of the solid-state fermented mixed grain increased from 41.95% to 89.66% and 8 kinds of essential free amino acids were increased 13 times compared to the raw materials. [This study suggested that solid-state fermentation using strain BA245 can be used for industrial applications due to their activity in production of grain enzyme-containing foods.]

C022

Effect of Metabolic Condition Medication and Probiotics Formulation on the Model Rats and Their Intestinal Microbiota

Seokcheon Song1, Joohyun Shin2, Jaegu Seo2, Myungjoon Jung2, and Eungbin Kim1*1Department of Systems Biology, Yonsei University, 2R&D Center, Cellbiotech, Co. Ltd.

Nowadays metabolic condition medications were consumed by patients in developed countries and some have side effect on intestine. It is known gut microbiota is important to intestinal health and related with diseases. Commercially available probiotics, Duolac-Gold (DG) was used to examine effects of the drugs since host diet can affect on shaping gut ecology. Two drug ingredients, leflunomide (LF), amlodipine (AMD), was chosen based on side effect profile, preliminary experiment. Here, we conducted in vivo experiment about effects of LF, AMD, DG on gut, gut microbiota with a rat model.Rats were distributed in 7 groups: control, LF-administered, LF plus low, high dose of DG-administered, AMD-administered, AMD plus low, high dose of DG-administered.Target probiotic, gram positive, negative microbes in stool were quantified. The probiotics decreased by LF, AMD but DG offset the effect of the drugs. LF, AMD decreased gram negative microbes but they were recovered by DG. In metabolic data, decrease of weight of rat by AMD was observed. Jejunum was inflated by AMD and it is a good agreement with reduced weight. Ileal ulcer by LF and AMD was also observed. All changes above by medications were alleviated by DG. To examin ulcer closely, cytokines in intestinal tissue were measured. They increased by LF, AMD but DG offset their increases. To conclude, LF, AMD can disturb gut microbiota but DG alleviates the effects of the drugs.[Supported by grants from Cellbiotech]

C023

Combination of Synthetic Genetic Circuitry and Microfluidics for Highly Sensitive Heavy Metal Ion Biosensors

Hyun Ju Kim1,2, Ji Won Lim3, Haeyoung Jeong1,4, Sang-Jae Lee5, Dong-Woo Lee6, Taesung Kim3,7, and Sang Jun Lee1,2*1Biosystems & Bioengineering Program, University of Science and Technology (UST), 2Microbiomics and Immunity Research Center, Korea Research Institute Bioscience & Biotechnology (KRIBB), 3Department of Biomedical Engineering, Ulsan National Institute of Science & Technology (UNIST), 4Superbacteria Research Center, KRIBB, 5Major in Food Biotechnology, Silla University, 6School of Applied Biosciences, Kyungpook National University, Daegu, Republic of Korea, 7Department of Mechanical Engineering, UNIST

The development of efficient microbial biosensors is essential for proper integration with micro/nanotechnologies. In order to produce whole-cell heavy metal biosensors with improved functions, we must select suitable sensory bio-parts and regulatory modules from an enormous quantity of genome information; these components can then be recombined to create synthetic genetic circuits. In this study, multiple copies of a cadC homolog encoding a heavy metal-responsive transcription factor were found in the genome of Bacillus oceanisediminis 2691, and the heavy metal specificities of the encoded proteins were characterized using a fluorescence reporter assay. For intracellular signal amplification, we constructed CadC- controlled T7 RNA transcription systems. As a result, cadmium and lead ions could be recognized specifically by CadC-T7 biosensors. Next, a chemostat-like microfluidic platform was combined with microbial cells to generate heavy metal biosensor devices with higher sensitivity. Our study shows the successful development of heavy metal biosensor microfluidic devices by integration of synthetic microbiology and nano-biotechnology [This work was supported by Next-Generation BioGreen 21 Program, RDA, Korea].

C024

Lactobacillus plantarum Suppressed ß-hexoaminidase, Histamine, and Expression of TNF-α and IL-4 in BPA-stimulated RBL-2H3 Cells

Jin A Lim and Sejong Oh*

Division of Animal Science, Chonnam National University of Korea

This study inspected the inhibitory effect of the glycoprotein isolated from Lactobacillus plantarum on bisphenol A tempted allergic inflammatory reaction in RBL-2H3 cells. RBL-2H3 cells were treated with Bisphenol A (50 μmol/ml) and the glycoprotein isolated from L. plantarum (5–100 µg/ml) for 30 min. histamine release, β-hexosaminidase and intracellular Ca2+ level were estimated in the medium. Activities of PKC, iNOS were measured by western blotting. Also IL-4 and TNF-α were measured by RTq-PCR. In this study, glycoprotein isolated from L. plantarum inhibited the histamine release, β-hexosaminidase, the intracellular Ca2+ level, and activity of iNOS in bisphenol-A treated RBL-2H3 cells. The results indicated that the glycoprotein isolated from L. plantarum inhibits expression of cytokines associated with allergy such as TNFα and IL-4 on the degranulation of mast cell. In conclusion, the results showed that degranulation, histamine release were inhibited by the glycoprotein of L. plantarum in the BPA stimulated RBL-2H3 cells, while amount of Ca2+ and iNOS were diminished. TNFα and IL-4, two of allergy-related cytokines were suppressed. Taken together might help to prevent allergy diseases induced by environmental hormone such as BPA.


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C025

Total Protein Isolated from Lactobacillus plantarum Has a Protective Character from Cadmium Chloride-stimulated Raw 264.7 Cells

Miyoung Shin and Sejong Oh*

Division of Animal Science, Chonnam National University

The food and water we consume are often contaminated with a range of chemicals and heavy metals, such as lead, cadmium, arsenic, chromium, and mercury that are associated with numerous diseases. Especially, cadmium is well known as one of the major components can cause a number of lesions in many organs. However, the mechanism of cadmium-toxicity and its inhibition are not clear. In this study, we demonstrated that total protein isolated from lactobacillus plantarum protects raw 264.7 cells from expression of inflammation-related factors stimulated by cadmium chloride (100 µM). Furthermore, we evaluated the cytotoxicity of cadmium using MTT assay and intracellular Ca2+ using fluorescence, and activities of activator protein PKC-α, iNOS, (AP)-1, MAPK, PCNA, and cyclin D1/CDK4 using immunoblot. The results obtained from this experiment indicated that total protein isolated from L. plantarum inhibits the intracellular Ca2+ mobilization. Also, it significantly suppressed inflammatory factors [expression of AP-1 (c-Jun and c-Fos), MAPKs (ERK, JNK and p38), iNOS]. With regard to cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly suppressed by treatment with total protein isolated from L. plantarum in the presence of Cd. Taken together, the findings suggested that total protein isolated from L. plantarum might be used as a food component for protection of inflammation caused by cadmium ion.

C026

Nexus Role of Paracoccus denitrificans in Simultaneous Removal of Nitrate, Iron and Arsenic

Sunhwa Park and Hor-Gil Hur*

School of Environmental Science and Engineering, Gwangju Institute of Science and Technology

Nitrate and arsenic are considered as drinking water contaminants and have been reported to co-exist in groundwater in various area around the world. Due to their toxicity, co-contamination of arsenic and nitrate can threat to health and environments. Therefore, technology for simultaneous removal of nitrate and arsenic is desirable because of co-existence of arsenic and nitrate in drinking water sources. Here, we demonstrated that denitrifying bacteria, Paracoccus denitrificans strain ATCC 17741 could effectively immobilize arsenic during biogenic nitrite-mediated Fe(II) oxidation. During this process, the nitrate, iron and arsenic were completely removed from the aqueous phase of culture medium and arsenic was adsorbed into goethite formed by P. denitrificans during 7 days incubation. Based on the sequential chemical extraction, high concentrations of arsenic was determined to be incorporated into the biogenic goethite (30.5 and 27.5% for As(III) and As(V), respectively). The results of this study emphasized that P. denitrificans can effectively sequester arsenic by the formation of goethite coupled with the consumption of both nitrate and iron and more arsenic was also incorporated into the crystalline goethite. Therefore, these result could be the one of potential strategies for simultaneous arsenic immobilization during Fe(II) oxidation and applied for concurrent removal of arsenic and nitrate remediation treatment in anoxic soils and groundwater.

C027

Determination of Antiviral Effects of Adenosine Analogues on Epstein-Barr Virus Infection

Miyeon Cho, Seok-Won Jung, Soomin Lee, and Hyojeung Kang*

College of Pharmacy and Institute of Microorganisms, Kyungpook National University

Cordyceps species are known to produce numerous active components and are used for diverse medicinal purposes because of their varied physiological activities, including their anticancer, anti-inflammatory, and antimicrobial effects. Cordycepin, one of adenosine analogues, differs from adenosine in that its ribose lacks an oxygen atom at the 3' position. Cordycepin has been reported to be a main effector molecule in Cordyceps extracts that executes antiviral activities against several viruses including influenza virus, plant viruses, human immunodeficiency virus (HIV), murine leukemia virus, and Epstein-Barr virus (EBV). In addition, adenosine and its deaminase inhibitor showed strong antiviral potentials that were about 4,000 times more potent than the activity of the direct inhibitory effect by adenine arabinoside on Herpes simplex virus (HSV)-1, suggesting adenosine itself plays an important role to produce antiviral activities. In this study, we show that cordycepin and adenosine possess antiviral and antitumor activities against EBV and EBV-associated gastric carcinoma, respectively. Furthermore, we report epigenetic mechanisms used by cordycepin and adenosine to exert their antiviral and antitumor effects. [This study was supported by grants from KRF (2015R1D1A3A01020679).]

Poster

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D001

Effect of Sub Minimal Inhibitory Concentration Chlorhexidine on Binding Characteristics of Streptococci and Actinomycetes

So Yeon Lee and Si Young Lee*

Department of Oral Microbiology, Colleage of Dentistry, Gangneung-Wonju National University

Chlorhexidine has been used for a long time as mouth washes for the control of dental caries, gingivitis and dental plaque. Minimal inhibitory concentration (MIC) is the lowest concentration of antimicrobial substance to inhibit the growth of bacteria. The concentrations lower than the MIC are called as sub minimal inhibitory concentrations (sub-MICs). Many studies have reported that sub-MICs of antimicrobial substances can inhibit virulence factors of bacteria. The aim of this study was to investigate the effect sub-MIC of chlorhexidine on biofilm formation by each bacterial strains and coaggregation of Streptococcus gordonii, Streptococcus mutans, Actinomyces naeslundii and Actinomyces odontolyticus. The biofilm formation of S. gordonii, A. naeslundii and A. odontolyticus was not affected by sub-MIC chlorhexidine. However, the biofilm formation of S. mutans was increased after incubation with sub-MIC chlorhexidine. Coaggregation of A. naeslundii with A. odontolyticus has been reduced by sub-MIC chlorhexidine. Coaggreagation of A. naeslundii with S. gordonii had not affected. These results indicate that sub-MIC chlorhexidine could influence on the binding properties such as biofilm formation and coaggregation in Streptococci and Actinomyces.

D002

Whole Genome-scale Transcriptomic Analysis of c-di-GMP Signaling in Enteropathogenic Escherichia coli

Hyung Tae Lee, Dalmuri Han, June Bong Lee, Chong-Hae Hong, and Jang Won Yoon*

College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University

Enteropathgenic Escherichia coli (EPEC) is in the family of Enterobacteriaceae that often cause diarrheal diseases of young children in developing countries. The main routes of EPEC transmission are consumption of the contaminated food, water, or fomites as well as direct contact with infected animals. EPEC can sense and respond to various environmental cues by signaling through the small molecules. Cyclic diguanylate (c-di-GMP) is one of such signaling molecules present in bacteria, but absent in eukaryotes or archaea, which modulates several cellular functions. However, the role of c-di-GMP signaling in the pathophysiology of EPEC has not been fully understood. Here we investigated the role of c-di-GMP signaling in the EPEC prototype strain, E2348/69, using genome-wide transcriptomics by RNA sequencing. To this end, both YaiC (a diguanylatecyclase) and RocR (a phosphodiesterase) were overexpressed in E2348/69 under the control of arabinose to manipulate intracellular c-di-GMP levels, which were previously characterized as c-di-GMP synthetase and hydrolase, respectively. Our experimental analyses revealed a global regulatory role of c-di-GMP in EPEC E2348/69, which involves in flagella motility, flocculation, biofilm formation, and virulence expression. These results imply that c-di-GMP signaling is important for the pathophysiology of EPEC. [This study was supported by a National Research Foundation Grant (NRF-2011-0010224) funded by Korean government, Republic of Korea.]

D003

Ferroportin Promote ROS Influx into Salmonella Containing Vesicle Resulting in Intramacrophage Bacterial Killing

Daejin Lim, Hyun-Ju Kim, Jea-Ho Jeong, Kwangsoo Kim, Kun-Hee Kim, Kyeongil Park, Shinan Li, and Hyon E. Choy*

Department of Microbiology, Chonnam National University Medical School

In response to microbial infection, defensin-like peptide hepcidin is induced to limit the serum ironby reducing iron release from macrophages through interleuklin-6 (IL-6) signaling.Here, we reportthat a nuclear receptor ERRγ mediates Salmonella infection through host iron alteration by hepcidininduction, and demonstrate an antimicrobial effect of ERRγ inverse agonist. Hepatic ERRγ geneexpression was induced by Salmonella-stimulated IL-6 signaling, resulting in an induction of hepcidin, and degradation of ferroportin on macrophage cell membrane, and eventual hypoferremia in mice. An inverse agonist of ERRγ restored the Salmonella-mediatedhypoferremia through reduction of ERRγ-mediated hepcidin gene expression, and exerted a potentantimicrobial effect on the Salmonella infection, thereby improving host survival. Further study indicated that mechanism underlying antimicrobial effect of inverse agonist of ERRγ was to induce ROS production in Salmonella Containing Vesicle by modulating iron content therein, consequence of which was enhanced bacteria killing.[This work was supported by NRF-2014R1A2A1A10051664.]

D004

Inhibition of Varicella-zoster Virus Replication by the Extract from Elaeocarpus sylvestris

Na-Eun Kim, So-Hee Bae, June-Eun Kim, and Yoon-Jae Song*

Department of Life Science, Gachon University

Varicella-Zoster virus (VZV), which is a member of the α-herpesvirus subfamily, causes varicella (chickenpox) by primary infection and reactivates to cause herpes-zoster (shingles). To identify a new anti-VZV drug candidate or develop a substitute for existing medicines, seventy percent ethanol extracts from plant materials were screened for their inhibitory activities on VZV replication in vitro. Crude ethanol, EtOAc fraction and hot water extracts of Elaeocarpus sylvestris leaves (ESE) distinctly inhibited the replication of the VZV pOka strain without showing any significant cytotoxic effect against MRC-5 cells. Furthermore, ESE considerably down-regulated VZV immediate early (IE) gene expression. Thus, ESE has a promising antiviral activity against VZV by reducing VZV IE gene expression and replication. [This research was supported by Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (No. 311063-5).]


34 | www.msk.or.kr

D005

The Quorum Sensing-dependent Factor(s) Can Modulate the Activity of Protease IV, a Major Virulence Factor of Pseudomonas aeruginosa

Jungmin Oh1,2,3, Soo-Kyung Kim1,2,3, Xi-Hui Li1,2,3, and Joon-Hee Lee1,2,3*1Lab of Microbiology, 2College of Pharmacy, 3Department of Pharmacy, Pusan National University

Pseudomonas aeruginosa can infect plants and animals, including humans. In humans, it can cause several disease on various body organ, such as the corneas the lungs and the kidneys. The major factor of the Pseudomonas corneal infection is the Protease IV (PIV). PIV is a quorum sensing (QS)-regulated exoprotease and a key virulence factor against Tenebrio molitor, an insect. PIV expressed in QS mutant (MW1, lasI‾,rhlI‾ double mutant of P. aeruginosa) showed much reduced activity. The cell-free culture supernatant (CFCS) from the PIV-overexpressing WT strongly induced the melanization of T. molitor larva, but the CFCS from the PIV-overexpressing MW1 rarely induced the melanization, even though it contained comparable amount of PIV to CFCS of WT. PIV is the lysyl endopeptidase, so we compared the activities of PIV-overexpressing in WT and MW1 using chromogenic substrate {N-(p-Tosyl)-Gly-Pro-Lys 4-nitroanilide acetate salt}. PIV-overexpressing WT showed strong PIV activity, but PIV-overexpressing CFCS in MW1 was not. When we purified PIVs from WT and MW1, the activity of PIV purified from MW1 was significantly lower than PIV from WT. Interestingly, when we add CFCS of piv mutant to the PIV purified from MW1, the activity of PIV from MW1 was restored to the level of the PIV from WT. We therefore suggest that there are some QS-dependent factors that modulates the Protease IV activity.[Supported by grants from KRF]

D006

A Salmonella Virulence Protein Interacts with PhoR That Activates Pho-regulon

Soomin Choi

Microbial Genetic Laboratory, College of Life Science, Kyung Hee University

Several intracellular pathogens, including Salmonella enterica require the virulence protein MgtC to survive within macrophages and to cause a lethal infection in mice. We now report that MgtC activates Pho-regulon including PhoB-PhoR two component system that expressed Phosphate regulator gene. PhoR is a histidine kinase sensor protein that appears to respond to periplasmic orthophosphate. PhoR respond phosphate from periplasm, auto-phosphorylate to histidine, and phosphorylate response regulator protein PhoB, then phosphate regulator gene has been activated. We establish that MgtC interacts with the a subunit of the Pho-regulon, PhoR, activated phosphorylation of gene regulator protein, PhoB.

D007

Eukaryotic Stress Response Gene ATF3 Provides Protection from Staphylococcus aureus and Listeria monocytogenes Infections

Sung-Yoep Lee, Suhkneung Pyo, and Dong Kwon Rhee*

School of Pharmacy, Sungkyunkwan University

Activating transcription factor (ATF)-3 is a stress-induced transcriptional regulator. The role of ATF3 in cancer has been well defined, but how ATF3 functions in bacterial infection is not well understood. Pneumococcal infection has been shown to induce ATF3 expression, which subsequently enhances cytokine production and provides protection from lethal Streptococcus pneumoniae infection, but the role of ATF3 in other Gram-positive (G+) infections remains unclear. Here, we report that infection with other G+ bacteria (Staphylococcus aureus and Listeria monocytogenes) and with G- bacteria (Escherichia coli as a negative control) also significantly induced ATF3 expression. Moreover, the production of cytokines (tumor necrosis factor alpha [TNF]-α, interleukin [IL]-1β, IL-6, and interferon [IFN]-γ) was enhanced by ATF3 in S. aureus and L. monocytogenes infection, but decreased in E. coli infection. In addition, in S. aureus and L. monocytogenes infections, ATF3 WT mice cleared bacteria more efficiently and had higher survival rates than ATF3 knockout (KO) mice. However, in E. coli infection, no significant difference was found in survival rate. Taken together, these data suggest that ATF3 provides protection from S. aureus and L. monocytogenes infections; however, the role of ATF3 in E. coli infection is more complicated and should be further elucidated. [Supported by NRF-2015R1 A2 A1 A10052511.]

D008

Isolation of New Bacteriophages to Control Pathogenic Bacteria, Bacillus cereus

Jeong-A Lim, Sojung Kim, Jonguk Kim, Jisoo Hong, Eunjung Roh, Kyu Seok Jung, Jae-Gee Ryu, and Jin-Woo Park*

Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration

Bacillus cereus is pathogenic bacteria that causes diarrhea and vomiting and contaminates various vegetables in addition to foods. Generally, sprouts were cultivated in hydroponics because of its advantages like even transmission of nutrient to plant. However, if some pollutants like bacteria were contaminated to media all of the sprouts in the same system also contaminated. In deed in our study, media contamination was connected to sprout contamination. After the seeds of Chinese cabbage were germinated and grown to sprouts, B. cereus (106 CFU/ml) was added to liquid media which was filled with sprouts roots. After 3 days, B. cereus was detected in surface-sterilized sprout body (104 CFU/ml). It means that B. cereus was internalized into the sprouts not only attached to. Proliferation of B. cereus in hydroponics aggravates the contaminated situation. In seed germination condition, the cell number of B. cereus in hydroponics was increased about 2 logs. To control B. cereus, biocontrol system using bacteriophage, a virus that infect and lyse host bacteria, can be adapted. We isolated six bacteriophages from soil samples. The bacteriophages showed strong lysis activity against B. cereus by reducing cell number above 6 logs. Most of 73 B. cereus strains isolated from vegetable were inhibited by bacteriophages. Taken together, we can conclude that bacteriophage can be applied to sanitation to control pathogenic bacteria in hydroponics. [This work was supported by grants from RDA.]

Poster

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D009

The Putative Fungicidal Molecules Show an Antifungal Effect on Candida albicans Virulence Through a Regulating the Mitochondrial Activity

Young Kwang Park1, Se Woong Kim1, Hwang Suk Kim2, Hee-Yoon Lee2, and Joon Kim1*1Lab. of Biochemistry, Division of Life Sciences, Korea University, 2Department of Chemistry, KAIST

The opportunistic pathogen Candida albicans colonizes the human skin and human mucosal surface. Undergoing morphogenesis from yeast to hyphal form in various environmental conditions is one of the well-known virulence factors of C. albicans. Small molecules were designed as chemical structural analogues of Bafilomycin which is a V-ATPase specific inhibitor. To characterize small molecules, we investigated the effect of small molecules on growth rate and the morphological transition. Growth and hyphal formation rate were decreased by treatment of small molecules. Moreover in the candidiasis murine model, molecule C has shown the effective antifungal activity. The microarray result which was using a set of RNA from control and drug-treated cells revealed mitochondrial related genes are influenced by molecule C. Also in 2D gel analysis data has shown the similar results as microarray data. As a conclusion from these results, molecule C has the antifungal effect and it appears to influence on mitochondrial activity. Further investigation on the mechanism will be presented in this study.

D010

Vacuoles are Essential for Morphogenesis and Virulence in Candida albicans

Se Woong Kim, Young Kwang Park, Yoo Jin Joo, Yu Jin Chun, Ju Yeon Hwang, Je-Hyun Baek, and Joon Kim*

Lab of Biochemistry, Division of Life Sciences, Korea University

The human opportunistic fungal pathogen Candida albicans undergoes a morphological transition under various conditions. Moreover, it is well known that morphogenesis is important for the onset of pathogenesis of C. albicans. In this study it is shown that filamentous growth critical for pathogenesis of C. albicans requires active intrinsic vacuole function. Using biochemical analyses, it was determined that the VMA4 and VMA10, putative E and G subunits of the vacuolar H+-ATPase complex, genes were overexpressed in a hyphal growing cell compared to that of a yeast growing cell. Deleting VMA4 or VMA10, both of which form a stalk domain in the complex, abolished intrinsic vacuolar functions, such as endosomal acidification and trafficking. Moreover, Vma4 and Vma10 were important for morphological conversion and hyphal-specific gene expression. These deletion mutants were also characterized as avirulent in mouse experiments. Furthermore, VMA4 and VMA10 deletion caused hypersensitive growth in the presence of fluconazole, suggesting that they are involved in the resistance to antifungal drugs. Based on these findings, roles were found for Vma4 and Vma10 of C. albicans in vacuole biogenesis, vacuole-dependent cytological processing, hyphal formation, as well as pathogenesis. These results suggest that the V-ATPase complex is a possible target for antifungal drug development in C. albicans.

D011

Ohmyungsamycins, New Antimycobacterial Cyclic Peptides, Activate Autophagy via AMP-activated Protein Kinase-mediated Signaling

Tae Sung Kim1,2, Yern-Hyerk Shin3, Hye-Mi Lee1,2, Soohyun Um3, Jin Kyung Kim1,2, Dong-Chan Oh3, and Eun-Kyeong Jo1,2*1Department of Microbiology, College of Medicine, Chungnam National University, 2Infection Signaling Network Research Center, 3Natural Products Research Institute, College of Pharmacy, Seoul National University

The AMP-activated protein kinase (AMPK) pathway activation is required for antimicrobial host defense against Mycobacterium tuberculosis (Mtb), a major pathogenic strain of human tuberculosis. During search for novel AMPK-activating agents with antimycobacterial effects, we had an attention on the newly identified Ohmyungsamycins (OMS) A and B, new cyclic peptides with antibacterial and anticancer effects. In this study, we show that OMS are essential autophagy activator leading to antimicrobial responses against Mtb, through activation of AMPK pathway in murine bone marrow- derived macrophages (BMDMs). OMS robustly activated autophagy induction and upregulated colocalization of LC3 autophagosomes with bacterial phagosomes in BMDMs. The activation of autophagy was required for OMS-induced antimicrobial responses against Mtb in BMDMs. We further showed that OMS triggered AMPK activation, which was essential for OMS-mediated autophagy activation. Collectively, these data show that OMS is a promising candidate for new antimycobacterial therapeutics through autophagy activation via AMPK dependent signaling.

D012

Antagonistics against Pathogenic Fusarium solani and Fusarium oxysporum by Novel Peptides from Bacillus amyloliquefaciens PT14

Hee Kyoung Kang1 and Yoonkyung Park1,2*1Department of Biomedical Science, Chosun University, 2Research Center for Proteineous Materials, Chosun University

Bacillus species have recently drawn attention due to their potential use in the biological control of fungal diseases. This paper reports on the antifungal activity of novel peptides isolated from Bacillus amyloliquefaciens PT14. Reverse-phase high-performance liquid chromatography revealed that B. amyloliquefaciens PT14 produces five peptides (PT14-1, -2, -3, -4a, and -4b) that exhibit antifungal activity but are inactive against bacterial strains. In particular, PT14-3 and PT14-4a showed broad-spectrum antifungal activity against Fusarium solani and Fusarium oxysporum. The PT14-4a N-terminal amino acid sequence was identified through Edman degradation, and a BLAST homology analysis showed it not to be identical to any other protein or peptide. PT14-4a displayed strong fungicidal activity with minimal inhibitory concentrations of 3.12 mg/L (F. solani) and 6.25 mg/L (F. oxysporum), inducing severe morphological deformation in the conidia and hyphae. On the other hand, PT14-4a had no detectable hemolytic activity. This suggests PT14-4a has the potential to serve as an antifungal agent in clinical therapeutic and crop-protection applications.


36 | www.msk.or.kr

D013

The Impact of Serum Albumin on Predation by Bdellovibrio bacteriovorus HD100

Ga Young Cho, Hansol Im, Ajay Monnappa, and Robert Mitchell*


Bdellovibrio bacteriovorus HD100 is predatory bacterium and is considered as a potential living antibiotic. As many bacteria can cause an infection within the blood, its components are important factors that may influence the predation activity. We show here, based upon predation patterns and microscopic analyses, that serum albumin binds to B. bacteriovorus HD100 and severely impacts the predation of E. coli.

D014

Transcriptomic Profiles from Bdellovibrio bacteriovorus HD100 during Predation on an Extended-Spectrum Beta-Lactamase (ESBL) Strain of Escherichia coli

SooIn Choi1, Mohammed Dwidar2, and Robert J. Mitchell1*1School of Life Science, Ulsan National Institute of Science and Technology2Okinawa Institute of Science and Technology, Japan

Bdellovibrio bacteriovorus is a Gram-negative bacterium considered to be a potential living antibiotic because of their unique predatory life and ability to attack a wide range of Gram-negative bacteria. In this regard, predatory bacteria are being considered as an alternative to conventional antibiotics against multi-drug resistant pathogens. In this study, we evaluated the predation mechanisms used by B. bacteriovorus HD100 while predating on an extended-spectrum beta-lactamases (ESBL) producing Escherichia coli via whole RNA-sequencing. [Supported by grants from DARPA]

D015

Genome-wide Transcriptome Analysis of Sch9-dependent Thermotolerance Mechanism Reveals the Dual Functional Heat Shock Factor 1, Hsf1, in Cryptococcus neoformansDong-Hoon Yang1, Kwang-Woo Jung1, Soohyun Bang1, Jang-Won Lee1, Min-Hee Song1, Yeonseon Lee1, Eunji Jeong1, Anna Floyd2, Richard Festa3, Giuseppe Ianiri4, Alex Idnurm4, Dennis Thiele3, Joseph Heitman2, and Yong-Sun Bahn1*1Department of Biotechnology, Center for Fungal Pathogenesis, Yonsei University, 2Departments of Molecular Genetics and Microbiology, Medicine, and Pharmacology and Cancer Biology, Duke University Medical Center, Durham 27710, NC, USA, 3Department of Pharmacology & Cancer Biology and Biochemistry, Medicine, and Phamacology and Cancer Biology, Duke University Medical Center, Durham 27710, NC, USA , 4Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, MO 64110, USA

Thermotolerance is a crucial virulence attribute for human fungal pathogen Cryptococcus neoformanss. Sch9 kinase suppresses C. neoformans thermotolerance, but its regulatory mechanism remains unknown. Here, we study the Sch9- dependent and -independent signaling networks modulating C. neoformans thermotolerance by using genome-wide transcriptome analysis. During temperature upshift, genes encoding for molecular chaperones and heat shock proteins are mainly upregulated, whereas those for translation, transcription, and sterol biosynthesis are highly suppressed. Notably, HSF1, encoding heat shock transcription factor 1, is downregulated during temperature upshift and SCH9 deletion suppresses its downregulation. Nevertheless, Hsf1 is essential for growth and its overexpression promoted C. neoformans thermotolerance. Transcriptome analysis using an HSF1 overexpressing strain supports the dual role of Hsf1 in the oxidative stress response and thermotolerance. Chromatin immunoprecipitation demonstrates that Hsf1 binds to the step-type like heat shock element (HSE) of its target genes more efficiently than to the perfect- or gap-type HSE in C. neoformans. This study not only provides further insight into the regulatory mechanism of C. neoformans thermotolerance, but also elucidates the regulatory mechanism of Sch9 in thermotolerence through a genome-scale identification of the Sch9-dependent genes.[Supported by grants from National Research Foundation of Korea (NRF).]

D016

Unravelling of the Target of Rapamycin (TOR1) Kinase Signaling Pathway in Human Fungal Pathogen Cryptococcus neoformans

Yee-Seul So1, Giuseppe Ianiri2, Alex Idnurm2, Jae-Hyung Jin3, and Yong-Sun Bahn3*1Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 2Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, MO 64110, USA, 3Department of Biotechnology, Center for Fungal Pathogenesis, Yonsei University

The TOR pathway has been implicated in regulating cellular responses to nutrients, including proliferation, translation, autophagy, and ribosome biogenesis. Here we identified two homologues of S. cerevisiae Tor, CNAG_ 06642 (Tor1) and CNAG_05220 (Tlk1, TOR-like kinase 1), in Cryptococcus neoformans. To study the TOR-1 signaling pathway, we attempt to construct the tor1Δ and tlk1Δ mutants and phenotypically analyze them. Although we fail to construct the tor1Δ mutant, we successfully construct the tlk1Δ mutant. The essentiality of TOR1 is independently confirmed by constructing the TOR1 promoter replacement strain by using a copper transporter4 (CTR4) promoter and the TOR1/tor1 heterozygous mutant in diploid C. neoformans strain background followed by sporulation analysis. To analyze the function of Tor1, we construct TOR1 overexpression mutant using a constitutively active histone H3 in C. neoformans. We find that Tor1 is involved in response to diverse stresses, including genotoxic stress, oxidative stress, thermo-stress, antifungal drug treatment, and production of melanin. To identify any TOR-related transcription factors, we screen C. neoformans transcription factor library that we constructed in our previous study and identify several potential downstream factors of Tor1. In conclusion, the current study provides insight into the role of the TOR signaling pathway in human fungal pathogens as well as C. neoforman.[Supported by grants form NRF]

Poster

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D017

Kinome Webs Reveal Novel Pathogenicity Networks in Human Fungal Pathogen Cryptococcus neoformans

Kyung-Tae Lee1, Yee-Seul So2, Dong-Hoon Yang2, Kwang-Woo Jung2, Jaeyoung Choi3, Dong-Gi Lee4, Hyojeong Kwon2, Juyeong Jang2, Li Li Wang2, Soohyun Cha2, Gena Lee Meyers2, Joohyeon Hong2, Soohyun Bang2, Je-Hyun Ji2, Goun Park2, Hyo-Jeong Byun2, Sung Woo Park2, Young-Min Park2, Gloria Adedoyin5, Taeyup Kim5, Anna K Averette5, Jong-Soon Choi4, Eunji Cheong2, Yong-Hwan Lee3, and Yong-Sun Bahn2*1Department of Biotechnology, Center for Fungal Pathogenesis, 2Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 3Department of Agricultural Biotechnology, Seoul National University, 4Biological Disaster Analysis Group, Korea Basic Science Institute, 5Department of Molecular Genetics and Microbiology, Medicine, and Pharmacology and Cancer Biology, Duke University Medical Center, USA

Cryptococcus neoformans is the leading cause of death by fungal meningoencephalitis; however, treatment options remain limited. To understand its underlying pathogenicity mechanisms and identify novel therapeutic targets, we have constructed 226 signature-tagged gene-deletion strains for 114 putative kinases and examined their phenotypic traits under 30 distinct in vitro growth conditions, in two different host environments. Phenotypic clustering analysis not only identified several novel kinases in known signalling pathways but also hitherto uncharacterized signalling cascades. Our large-scale virulence assays using an insect model and signature-tagged mutagenesis-based infectivity assays in a murine model discovered 60 pathogenicity-regulating kinases in diverse biological categories: growth and the cell cycle, nutrient metabolism, the stress response and adaptation, cell signalling, cell polarity and morphology, vacuole trafficking, tRNA modification, and other previously unknown functions. Our comprehensive fungal kinome analysis provides insights into the pathobiological signalling circuitry of C. neoformans and suggests novel anticryptococcal or antifungal drug targets.

D018

Detection and Growth Inhibition of Streptococcus mutans by Aptamers

kyeong-Ah Lee1, Simranjeet Singh Sekhon1, Gna Ahn1, Ji-Young Ahn1, Yang-Hoon Kim1, Sung-Jin Cho2, and Jiho Min3*1Department of Microbiology, 2Department of Biology, 3Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University

The Streptococcus mutans bacterial species plays a leading role in the initiation of dental caries. Among important virulence factors of this pathogen, its ability to form the biofilm is vital not only to its survival and persistence in the oral cavity, but also for its pathogenicity as well. Here we developed multifunctional aptamer for growth inhibition and detection of Streptococcus mutans. Aptamers are single-stranded oligonucleotides that interact with target molecules with high affinity and specificity. With advantages in targeted therapy and diagnosis, aptamers are considered as great potential ligand. We collected DNA aptamer that bind to S. mutans and developed the aptamer-based detection assay for specific detection of the cell. Moreover, several of aptamers inhibit cell growth of S. mutans more than control. This assay with high specificity can be used as an alternative method for the therapy and detection of oral disease. [This work was carried out with the support of "Cooperative Research Program for Agriculture Science & Technology Development (Project title: Risk Assessment Research and Development of Rapid Diagnostic Method for Biological, Chemical and Environmental Animal Disease, Project No: PJ01052301) Rural Development Administration, Republic of Korea. This work was supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education and National Research Foundation of Korea (NRF-2015H1C1A1035921).]

D019

Production and Characterization of Sodium Hydroxide Induced Vibrio parahaemolyticus Ghosts as a Potential Vaccine Candidate

Hyun Jung Park, Seongmi Ji, Nagarajan Vinod, Sung Oh, Jung Mo Koo, Han Byul Noh, Ki-Sung Lee, Sei Chang Kim, and Chang Won Choi*

Department of Biology & Medicinal Science, Pai Chai University

Vibrio parahaemolyticus PCU-1 ghosts (VPGs) were generated by chemically- induced lysis and the method is based on minimum inhibitiory concentration (MIC) of sodium hydroxide (NaOH), acetic acid (CH3COOH), boric acid (BH3O3), citric acid (C6H8O7), maleic acid (C4H4O4), hydrochloric acid (HCl) and sulphuric acid (H2SO4). The MICs of NaOH, CH3COOH, BH3O3, C6H8O7, C4H4O4, HCl, and H2SO4 were 3.125, 6.25, < 50.0, 25.0, 6.25, 1.56, and 0.781 mg/ml, respectively. Except CH3COOH and BH3O3, the lysis efficiency of V. parahaemolyticus cells was reached 100% at 10 min after treatment of other chemicals. Nevertheless, real-time PCR confirmed that NaOH only induced 100% DNA-free VPGs. Therefore, NaOH was selected as the best chemical to produce VPGs as a potential vaccine candidate. The formation of the trans-membrane lysis tunnel structure in NaOH induced VPGs, which was observed by scanning electron microscopy. SDS-PAGE and agarose gel electrophoresis supported that cytoplasmic proteins and genomic DNA released from NaOH induced VPGs to culture medium through the tunnel structure. In conclusion, the results of this study suggest that the NaOH induced VPGs are faster, more economical and safer method than protein E-mediated lysis cassette in inactivated vaccine research.[This work was supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education and National Reserch Foundation of Korea (2015035949).]

D020

Production and Characterization of Hydrochloric Acid Induced Listeria monocytogenes Ghosts (LMGs) as a Potential Vaccine Candidate

Seongmi Ji, Hyun Jung Park, Nagarajan Vinod, Sung Oh, Jung Mo Koo, Han Byul Noh, Ki-Sung Lee, Sei Chang Kim, and Chang Won Choi*

Department of Biology & Medicinal Science, Pai Chai University

Listeria monocytogenes ghosts (LMGs) were generated by chemically induced lysis and the method is based on minimum inhibitiory concentration (MIC) of sodium hydroxide (NaOH), acetic acid (CH3COOH), boric acid (BH3O3), citric acid (C6H8O7), maleic acid (C4H4O4), hydrochloric acid (HCl) and sulphuric acid (H2SO4). The MICs of NaOH, CH3COOH, BH3O3, C6H8O7, C4H4O4, HCl, and H2SO4 were 6.25, 6, 25, < 50, 25, 12.5, 6.25, and 12.5 mg/ml, respectively. Among those, the lysis efficiency of L. monocytogenes cells was reached 100% at 5 min after treatment of H2SO4, 10 min after treatment of NaOH, 15 min after treatment of HCl and 30 min after treatment of maleic acid. The formation of the trans-membrane lysis tunnel structure in respective chemicals induced LMGs, which was observed by scanning electron microscopy. SDS-PAGE and agarose gel electrophoresis supported that cytoplasmic proteins and genomic DNA released from H2SO4, NaOH and HCl induced LMGs, respectively, to culture medium through the tunnel structure. Nevertheless, real-time PCR confirmed that HCl only induced 100% DNA-free VPGs. Therefore, HCl was selected as the best chemical to produce VPGs as a potential vaccine candidate.[This work was supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education and National Reserch Foundation of Korea (2015035949).]


38 | www.msk.or.kr

D021

Identification and Mechanism of LL37 and Its Analogs with Potent Antimicrobial Activity against Acinetobacter baumannii Strains

Eunji Park1,2 and Yoonkyung Park1,2*1Research Center for Proteineous Materials (RCPM), 2Department of Biotechnology and BK21-Plus Research Team for Bioactive Control Technology, Chosun University

The emergence of multidrug-resistant Acinetobacter baumannii is recently becoming increasingly important as nosocomial infection and has affected people with compromised immune system. Thus, effective novel antimicrobial agents are urgently required. Cationic antimicrobial peptides (CAMPs) are thought to play an important self- defense role in many organisms. Because of their ability to disrupt the bacteria membrane, leading to cytoplasmic disruption and cell death, so AMPs provide an alternative to existing antibiotics. However, instability by proteases generated by human inherently restrict the AMPs. We therefore designed novel cationic antimicrobial peptides by substituting residues in truncated human cathelicidin LL37. The antimicrobial activities, anti-biofilm activities of the five antimicrobial peptides were tested against Acinetobacter baumannii. Moreover, these peptides were found to be resistant to proteolytic degradation, but did not completely exhibited hemolytic activity and were not cytotoxic to normal human keratinocytes except for LL37. LL37 and its analogs appeared α-helical or β- sheet structures within bacterial membranes by Circular dichroism analysis. Furthermore, we carried out bacterial killing and membrane penetration experiments whether LL37 and its analogs penetrated membrane of bacteria. Collectively, our finding indicated LL37 analogs may be attractive candidate antibiotic agents against multidrug-resistant Acinetobacter baumannii.

D022

Characterization of Antimicrobial Activity and Mechanism of Antimicrobial Peptide against Bacteria

Su Jin Ko1,2 and Yoonkyung Park1,2*1Research Center for Proteineous Materials (RCPM), 2Department of Biotechnology and BK21-Plus Research Team for Bioactive Control Technology, Chosun University

Bacterial drug resistance is a major problem in human health as it has led to the reduced efficacy of conventional antibiotics. Thus, the identification of novel antibiotics is essential. Antimicrobial peptides are found in various living organisms, including plant, insects, amphibians, and mammals, where the play important roles in defense. Antimicrobial peptide (AMP) have been increasing interest as alternative to conventional antibiotics due to their broad spectrum antimicrobial activity and reduced possibility for the development of bacterial drug resistance. A novel antimicrobial peptide named MAC was isolated from the venom of the solitary bee Macropis fulvipes. MAC exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, multidrug resistance bacteria strains and did not cause significant lysis of human red blood cells and were not cytotoxic to normal keratinocytes. The CD spectra of MAC measured in the presence of trifluoroethanol (TFE) and sodium dodecyl sulfate (SDS) showed a high content α-helices. Furthermore, results of NPN uptake assay and scanning electron microscopy (SEM) indicated that peptide killed microbial cells by increasing membrane permeability and damaging membrane. Collectively, the results suggest that peptide have potential for use as novel antimicrobial agents.

D023

Effect of HAMP and T1AMP on the Antimicrobial Activity against Pseudomonas aeruginosa and Multidrug-resistant Pseudomonas aeruginosa

Min Kyung Kim1,2 and Yoonkyung Park1,2*1Research Center for Proteineous Materials (RCPM), 2Department of Biotechnology and BK21-Plus Research Team for Bioactive Control Technology, Chosun University

Antimicrobial peptides (AMPs) are important molecules of the host defense system against invading pathogen. Antimicrobial peptides are small molecules containing amino acids 10 to 50 amino acid residues with positive charge (generally +2 to +10), which exhibit antimicrobial activity against Gram- negative bacteria, Gram positive bacteria, yeasts and fungi and viruses. In this study, antimicrobial peptide HAMP was identified from the scorpion Heterometrus petersii, which is an amphipathic α-helical structure and antimicrobial activity. Base on this parent peptide HAMP, we designed analogs peptide T1AMP by truncation and substitution. The analog peptide T1AMP have strong antimicrobial activity against Gram negative bacteria including Pseudomonas aeruginosa and multidrug-resistant Pseudomonas aeruginosa. Furthermore, these peptide a little hemolytic activity on red blood cell and low cytotoxicity of HaCaT cell. Taken together, the results of this study indicate that the HAMP and analog peptide T1AMP may have antimicrobial activity and effective therapeutic agent against multidrug- resistant Pseudomonas aeruginosa.

D024

Antimicrobial Activity, Anti-biofilm and Action Mechanism of Charge-enriched AMPs against Both Gram-positive and Gram-negative Bacteria with Therapeutic Potential for Clinical Antibiotic-resistant

Hyo Mi Han1 and Yoonkyung Park1,2*1Research Center for Proteineous Materials (RCPM), 2Department of Biotechnology and BK21-Plus Research Team for Bioactive Control Technology, Chosun University

Cationic antimicrobial peptides (AMPs) are essential components of the innate immune system, offering protection against invading pathogenic bacteria. In nature, AMPs serve as antibiotics with broad-spectrum antimicrobial and anti-biofilm properties. However, low effective stability in high-salt environments and physiological instability in biological membranes limit the applicability of naturally occurring AMPs as novel therapeutics. We therefore designed short synthetic cationic peptides by substituting key residues in myxinidin, an AMP derived from the epidermal mucus of hagfish, with lysine, arginine, and tryptophan. Moreover, these peptides showed high binding affinity for both lipopolysaccharides and lipoteichoic acids and inhibited biofilm formation by most bacteria, but did not cause significant lysis of human red blood cells and were not cytotoxic to normal human keratinocytes. In addition, bacterial killing and membrane permeation experiments demonstrated that the myxinidin analogs permeated through bacterial membranes, leading to cytoplasmic disruption and cell death. Taken together, these findings suggest myxinidin analogs may be promising candidate antibiotic agents for therapeutic application against antibiotic-resistant bacteria.

Poster

www.msk.or.kr | 39

D025

Efficacy of Antimicrobial Peptide on Antibacterial, Anti-biofilm Activity

Jong Gwan Park1,2 and Yoon Kyung Park2*1Department of Convergence Science, Kongju National University2Research Center for Proteineous Materials (RCPM), Chosun University

Dental caries and periodontitis are common bacterial mouth infections. Oral biofilms are multispecies microbial communities that exhibit high resistance to antibiotics. Antimicrobial peptides (AMP) are emerging as promising antimicrobial agents due to their rapid bactericidal activites. This study describes AMPs exhibits a broad spectrum of bactericidal activity with eradicating capability on oral pathogens and respective biofilm. It is also stable in saline solution, saliva, brain-heart infusion medium. We further compared the antimicrobial activity kinetics of AMP with chlorhexidine. Flow cytometry was used to test if antimicrobial peptide can permeabilize the membranes of oral pathogens. a significant proportion of oral pathogens treated with antimicrobial peptide displayed propidium iodide fluorescent signal and the number of the bacterial cells with fluorescent signal increased with the dose dependent manner. Additionally, these did not cause lysis of human red blood cell and were non cytotoxic to KB, pulp cell called epithelial cell and ondontoblast. This study provides a proof of concept in applying antimicrobial peptides in the clinical perspective.

D026

Antibacterial and Anti-inflammatory Activities of CMA3 Peptide with Low Cytotoxicity in Escherichia coli

Jong-Kook Lee1 and Yoonkyung Park1,2*1Research Center for Proteinaceous Materials (RCPM), 2Department of Biotechnology and BK21 Research Team for Protein Activity Control, Chosun University

CA-MA is a hybrid antimicrobial peptide (AMP) derived from two naturally occurring AMPs, cecropin A and magainin 2. CA-MA shows strong antimicrobial activity against Gram-negative and Gram-positive bacteria but also exhibits cytotoxicity toward mammalian cells. Our objective was to identify CA-MA analogues with reduced cytotoxicity by systematic replacement of amino acids with positively charged R groups, aliphatic R groups, or polar R groups. CMA3 appeared to act by inducing pore formation (toroidal model) in the bacterial membrane. In cytotoxicity assays, CMA3 showed little cytotoxicity toward human red blood cells or HaCaT cells. Additionally, no fluorescence was released from small or giant unilamellar vesicles exposed to 60 μM CMA3 for 80 s, whereas fluorescence was released within 35 s upon exposure to CA-MA. CMA3 also exerted strong lipopolysaccharide- neutralizing activity in RAW 264.7 cells, and BALB/c mice exposed to LPS after infection by Escherichia coli showed improved survival after administration of one 0.5-mg/kg of body weight or 1-mg/kg dose of CMA3. Finally, in a mouse model of septic shock, CMA3 reduced the levels of proinflammatory factors, including both nitric oxide and white blood cells, and correspondingly reduced lung tissue damage. This study suggests that CMA3 is an antimicrobial/ antiendotoxin peptide that could serve as the basis for the development of anti-inflammatory and/or antimicrobial agents with low cytotoxicity.

D027

Antibacterial and Anti-inflammatory Effects of Novel P-AMP against Human Pathogens

Na hee Kang1,2 and Yoonkyung Park1,2*1Research Center for Proteineous Materials (RCPM), 2Department of Biotechnology and BK21-Plus Research Team for Bioactive Control Technology, Chosun University

Numerous antimicrobial peptides (AMPs) from natural substance have been identified, isolated and characterized. We evaluated antimicrobial activities of P-AMP, a novel antimicrobial peptide, in vitro. The novel peptide shows strong antimicrobial activity against Gram-negative and Gram-positive bacteria but also exhibits no cytotoxicity toward mammalian cells. P-AMP appeared to act in the bacterial membrane. In cytotoxicity assays, P-AMP showed no cytotoxicity toward RAW 264.7. Additionally, binding of lipopolysaccharide to macrophages results in pro-inflammatory cytokine secretion. Then, P-AMP has strong lipopolysaccharide neutralizing activity in RAW 264.7 cells exposed to LPS of P. aeruginosa. The peptides were investigated for their ability to inhibit LPS-mediated cytokine release from RAW264.7 to bind LPS in solution, and when LPS is already bound to macrophages. Finally, we conclude that P-AMP is an antimicrobial and anti-endotoxin peptide that could serve as the basis for the development of anti-inflammatory and antimicrobial agents with low cytotoxicity. This study suggests that P-AMP acts as host defense molecules that exert antimicrobial effects by targeting the lipopolysaccharide of P. aeruginosa, Gram-negative bacteria. Moreover, it has been reduced production of pro-inflammatory mediators and correspondingly reduced pulmonary disease such as cystic fibrosis.

D028

Immunization of Mice with Irradiated Whole Cell Vaccine Confers a Significant Degree of Protection against a Lethal Infection of Streptococcus agalactiae

A-Yeung Jang, Yong Zhi, Bum Joo Kim, Zhao Lei, Zhang Jing, Shunmei Lin, and Ho Seong Seo*

Radiation Biotechnology Division, Korea Atomic Energy Research Institute

The Gram-positive bacterium group B Streptococcus (GBS), known as Streptococcus agalactiae, is the most common cause of meningitis in the newborns. It is also increasingly associated with invasive disease in non- pregnant adults, especially among the elderly and individuals with underlying chronic illnesses. Since GBS whole cell vaccine (WCV) is expected to confer protection against GBS infection, we investigated the efficacy of irradiated killed WCV as compared to chemically killed WCV. GBS WCV was prepared by treating gamma-irradiation at 9kGy/hr or formalin for 2hr. ICR mice were immunized through intraperitoneal injection. Although irradiated WCV increased anti-GBS Ab titer in serum higher than level to chemical WCV, it was elicited significantly higher rate of survival in GBS infection model. Also, irradiated WCV-vaccinated mice were increased Ab titer to other serotype GBS strains compare with chemical WCV. Moreover, splenocytes isolated from irradiated WCV-vaccinated mice were increased population of CD3+ CD4+ T cells than chemical WCV and it was increased level of CD4+ T cell-associated cytokine (TNF-α, IL-6 and IL-10) with chemical WCV. These findings suggest that irradiated WCV elicit effective response of CD3+ CD4+ T cells that protects GBS infection.


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D029

Pathogenicity of Newly Isolated Human Ileal Streptococci

Dong-Wook Hyun, Min-Soo Kim, Tae Woong Whon, Na-Ri Shin, Mi-Ja Jung, Pil Soo Kim, Hyun Sik Kim, June-Young Lee, Woorim Kang, and Jin-Woo Bae*


Two novel isolates, designated strain I-G2 and I-P16, were isolated from ileal fluid of human who had undergone ileostomy by reason of colorectal cancer. The isolates were assigned to the members in mitis group streptococci and identified as novel species closely related with S. parasanguinis and S. australis based on polyphasic analyses. Thus, we designate the two isolates as Streptococcus ilei. To assess abundance and incidence of S. ilei in human body sites, we estimated S. ilei frequencies in various human body sites using metagenomic data (506 experiments, 6,228 samples and 22,875,147 reads) from Human Microbiome Project (HMP) database with matching S. ilei specific sequence. S. ilei occurs with 0.18 to 2.09% relative abundance and 19 to 86.98% incidence depending on each body site. To identify pathogenic potential of S. ilei, cytotoxicity of S. ilei was ascertained by MTT assay on A549 and HT-29 cell lines. S. ilei and its supernatant show cytotoxic response to A549 and HT-29 cell lines as similar as pathogenic S. pneumoniae. Furthermore, exposures of S. ilei via intraperitoneal routes causes mortality in immunocompromised mice and healthy mice models. Thus, these results disclose presence of S. ilei in human microbiota and demonstrate its pathogenic potential. [Granted by Mid-career Researcher Program (2012-0008806) through the National Research Foundation of Korea.]

D030

Prevalence of Respiratory Viruses in Patients with Acute Respiratory Infections in Jeonbuk, 2015

Chan mun Jin1, Yeun Jeong Kim1, Keung Eu No1, Seouk Hyeon Lim1, Cheon Hyeon Kim1, Hee Dong Jung2, Hyang Min Cheong2, Sung Soon Kim2, and Ki Soon Kim2*1Jeollabukdo Institute of Health and Environment Research, 2Center for Infectious diseases, KNIH, KCDC

In this study, to investigate the viral pathogen causing Acute Respiratory Infections (ARI), multiplex PCR/RT-PCR was performed on respiratory specimens (throat or nasal swab) collected from the ARI patients. And the statistical analysis was performed to investigate the characteristics of age distribution, seasonality, and clinical features of ARI patients with respiratory virus infection in Jeonbuk, 2015. We performed multiplex PCR/RT-PCR on respiratory specimens to determine the prevalence of 16 viruses including adenovirus(HAdV), parainfluenza virus(HPIV) 1, 2, 3, respiratory syncytial virus(HRSV) A and B, influenza virus (IFV) H1N1, H3N2, B, human coronavirus(HCoV) 229E, NL63 and OC43, human rhinovirus(HRV), human bocavirus(HBoV), human metapneumovirus (HMPV) and Enterovirus(HEV). Respiratory viruses were detected by 51.3% of enrolled patients (n=779) in 2015. The most common respiratory virus isolated was influenzavirus 82 cases (23.4%) followed by rhinovirus 145 cases (41.3%), enterovirus 49 cases (6.3%), adenovirus 44 cases (12.5%), parainfluenzavirus 33 cases (9.4%), coronavirus 18 cases (5.1%), metapneumovirus 12 cases (3.4%), respiratory syncytial virus 10 cases (2.8%) and bocavirus 7 cases (2.0%). A large proportion of respiratory infections in the community in Jeonbuk was associated to viruses. HRSV, IFV, and HCoV infections peaked on winter season, in summer, HEV were epidemic.[Supported by grants from KCDC]

D032

Chitosan Nanoparticle Supplemented Diet Alters the Zebrafish Gut Microbiota

Chathurica Udayangani, Sajith Dananjaya, Seung Beom Seo, and Mahanama De Zoysa*

College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungnam National University

This study was conducted to investigate the effects of dietary CNP on the gut microbial communities of zebrafish. Fish diet with CNPs was prepared by adding 2% of CNP to the commercial feed on a weight basis. Zebrafish (Danio rerio) were maintained (12 fish/tank) for 4 weeks and fecal matter was collected. 16S rRNA gene sequence based metagenomics sequencing was conducted to understand the change of microbiome profiles. Total of 56 and 53 operational taxonomic units (OTUs) were resulted for control and CNP treated groups, respectively. Upon CNP treatment considerable changes in the natural gut bacterial composition was observed. In both control and CNP treatments, phylum Proteobacteria was found to be the most abundant in zebrafish gut as reported in previous studies. However, 19% reduction in phylum Proteobacteria and 82.02% increase in phylum Fusobacteria were observed in the fish fed with CNP supplemented diet. Even though the exact function of certain microbial species are yet to be discovered, the results suggest that CNP could cause beneficial modifications of gut microbiome to enhance the digestibility and immunity of fish.

Keywords: Chitosan nano-particles (CNP); gut; Metagenomics; Proteobacteria; Fusobacteria; zebrafish

[This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2014R1A2A1A-11054585).]

D033

Fusarium oxysporum Infestation of Zebrafish in Laboratory System

Sang Yeop Shin, Chanuka Kulatunga, Dong Joon Kim, Bae Keun Park, and Mahanama De Zoysa*

College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungnam National University

Fusarium oxysporum is a highly diverse fungi inhabited in every ecosystem as a saprophyte or a pathogenic fungi. Recently, unusual F. oxysporum infestation was identified from zebrafish (Danio rerio) culture system. Initially, rapid whitish smudge was appeared in the water with the severity, fungal blooming on fish tank walls, clogging of water circulatory tubes was observed. Reduction of fish vigor and fungal gill infection of zebrafish was noticed in the affected water systems. Fungal isolation was done by inoculating the specimens on potato dextrose agar (PDA) at room temperature. Microscopic studies on fungal hyphae in the water system and the PDA, colony pigmentation, clamidospore formation and the presence of macro and microspores were morphologically confirmed the fungi as the F. oxysporum. Furthermore, the internal transcribed spacer regions of nuclear rDNA repeat units (ITS) sequences of isolated fungi were identical to nine F. oxysporum sequences in Genbank. Experimental hypodermis injection of zebrafish with F. oxysporum was developed the infection by revealing the virulence of these isolates. Histopathological studies were carried out to examine the pathogen invasion in zebrafish tissues.

Keywords: Danio rerio; Fusarium oxysporum; infestation; fungal hyphae

[This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2014R1A2A1A-11054585).]

Poster

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D034

Antibiotic Resistance is Induced by a Bacterial Starvation Signal in Vibrio cholerae

Hwa Young Kim, Young Taek Oh, and Sang Sun Yoon*

Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine

When bacteria encounters variety of growth-inhibitory stresses, the stringent response (SR) is activated by accumulation of (p)ppGpp, a small nucleotide regulator known as a stress alarmone. Accumulation of (p)ppGpp results in a profound reprogramming of global gene expression. Here, we show that the bacterial capability to produce (p)ppGpp is involved in regulating antibiotic resistance in V. cholerae. N16961, a 7th pandemic V. cholerae strain, became resistant to tetracycline, when (p)ppGpp accumulation was induced. N16961 and ΔrelAΔspoT, a (p)ppGpp-overproducing mutant strain, were more resistant to the treatment of lethal concentration of tetracycline, when compared with (p)ppGpp0 ((p)ppGpp-null mutant). Similar effects were also induced, in response to the treatment with other antibiotics, such as erythromycin and chloramphenicol. Furthermore, the ΔrelAΔspoT mutant, which mounted resistance, lost typical curved-rod shape morphology and looked smaller in size based on scanning electron microscope analysis. N16961, when harvested at stationary phase, also exhibited similar cell shape change, while (p)ppGpp0 mutant maintained their normal cell shape. We also found that N16961 gained smaller cell shape phenotype, when (p)ppGpp accumulation was induced by serine hydroxamate at exponential phase. Together, our results demonstrate that the alteration of bacterial cell phenotype by accumulation of intracellular (p)ppGpp is associated with antibiotic resistance in V. cholerae.

D035

Structural Insight of Substrate Promiscuity for 2-deoxyribose-5-phosphate Aldolase (DERA) from Streptococcus suis

Thinh-Phat Cao1, Joong-Su Kim2, and Sung Haeng Lee1*1Department of Cellular and Molecular Medicine, Chosun University School of Medicine, 2Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology

2-deoxyribose-5-phosphate aldolase (DERA) belongs to a class I aldolase catalyzing aldol condensation of two aldehydes in the active site, and is particularly intersting target to drug manufacture. Structural and biochemical studies have shown that the active site of DERA is typically flexible and displays broader substrate specificity. The most distinctive structural feature of DERA is short and flexible C-terminal region, which is also responsible for substrate recognition. Therefore, substrate tolerance may be related to the C-terminal structural features of DERA. Here, we determined the crystal structures of full length and C-terminal truncated DERA from Streptococcus suis (SsDERA). In common, both contained the typical (α/β)8 TIM-barrel fold of class I aldolases. Interestingly, C-terminal truncation resulting in missing the last α9 and β8 secondary elements, allowed DERA to maintain activity comparable to the full-length enzyme. Typically, Arg186 and Ser205 residues at the C-terminus appeared mutually supplemental or less indispensible for substrate phosphate moiety recognition. Our results suggest that DERA might adopt a shorter C-terminal region than conventional aldolases during evolution pathway, resulting in a broader range of substrate tolerance through active site flexibility.

D036

Identification of Essential Genes of Pseudomonas aeruginosa for It Is Growth in Airway Mucus

Mohammed Mohammed and Sang Sun Yoon*

Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine

Pseudomonas aeruginosa has been identified as an important causative agent for airway infection. Mucin is a major component of airway mucus and is heavily O-glycosylated with a protein backbone. Airway infection is known to be established with bacterial adhesion to mucin. However, genes involved in mucin degradation or utilization still remains elusive. In this study, we thought to provide a genetic basis of P. aeruginosa airway growth by identifying those genes. First, we compared genome-wide expression profiles of PAO1, a prototype strain, grown in M9-mucin (M9M) and M9-glucose (M9G) media by RNASeq analyses. PA2939 and PA0122 genes encoding putative aminopeptidase and a hemolysin homologue were among top 5 genes, whose expression were increased >100-fold in M9M-grown PAO1. Second, a PAO1 transposon (Tn) insertion mutant library was screened for mutants defective in growth in M9M media. Six mutants with Tn insertion were determined to exhibit faulty growth in M9M. These genes are involved in the synthesis of leucine and tryptophan. Importantly, all of these mutants were incapable of establishing mouse airway infection, suggesting that these gene functions are required for P. aeruginosa in vivo infectivity. Further mechanistic dissection of this particular process will reveal new drug targets, inhibition of which could control recalcitrant P. aeruginosa infections.

D037

Anti-tumor Effect of Quercetin in EBV-associated Human Gastric Carcinoma

Hwan Hee Lee, Seulki Lee, Hyojeong Kang, and Hyosun Cho*

Department of Pharmacy, Duksung Women’s University

Licorice extracts have been widely used in herbal and folk medications. Glycyrrhiza yields diverse range of biological compounds including triterpene (glycyrrhizin, glycyrrhizic acid) and flavonoids (quercetin, liquiritin, liquiritigenin, glabridin, licoricidin, isoliquiritigenin). The flavonoids of licorice are known to have strong activities against cancer. Quercetin, the most abundant compound in flavonoids, has been shown to perform anti-ulcer, anti-cancer, antioxidant and anti-inflammatory properties. Latent infection of EBV leads to serious malignancies such as Burkitt’s lymphoma, Hodgkin’s disease and gastric carcinoma (GC). In fact, EBVaGC is known to be one of the most common cancers among EBV associated tumors.In this study, we first examined anti-cancer effect of quercetin and isoliquiritigenin in vivo xenograft mouse models implanted with EBV negative carcinoma (MKN74 cells) as well as EBV positive carcinoma (SNU719 cells). Secondary, we explored the molecular mechanisms responsible for the anti-viral and anti-cancer activities. The results showed that quercetin and isoliquiritigenin have the effect of anti-tumor in mice injected with or without EBV. Also, our results indicated that quercetin affects the expression of p53-associated proteins including p21, caspases and Bcl-2 families as well as the expression of EBNA-1, BZLF-1, and LMP-1 proteins in tumor tissue specimen of mice injected gastric carcinoma of presence or absence with EBV.


42 | www.msk.or.kr

D038

The Anti-HSV Effect of Quercetin in Raw 264.7 Cells via Downregulation of Inflammatory Responses

Seulki Lee, Hwan Hee Lee, and Hyosun Cho*

Department of Pharmacy, Duksung Women’s University

Quercetin is one of major ingredients derived from Glycyrrhiza uralensis, which is largely used as traditional medicine in Asian countries. Quercetin was reported to have several biological activities including anti-viral effect as well as anti-inflammatory effect. We explored the molecular mechanism that links anti-viral and anti-inflammatory activities using HSV infection system.To identify the downregulation of inflammatory response, raw 264.7 cells were treated with various concentrations of quercetin with or without HSV infection for 24 h and cell pellet (or supernatant) was collected. Then, we performed HSV plaque assay and ELISA assay. Next, we measured the expressions of HSV proteins (ICP0 and gD), NFkB and TLRs associated with anti-inflammatory genes using qPCR and Western blot analysis.Quercetin significantly decreased HSV infectivity in raw 264.7 cells through HSV plaque assay, and also suppressed the production of inflammatory cytokines (IL-1β and TNF-α). In addition, quercetin decreased the expression of HSV proteins (ICP0 and gD) and inflammatory genes including NFkB and TLR3 in raw264.7 cells. Therefore, we suggest that the anti-viral effect of quercetin was directly correlated with anti- inflammatory responses in HSV-infected raw 264.7 cells.

D039

KSHV Infection of Primary Human Endothelial Cells Induces Complement Activation by Exosome-mediated Binding of Properdin

Hyungtaek Jeon1, Jisu Lee1, Seung-min Yoo1, Shou-Jiang Gao2, and Myung-Shin Lee1*1Department of Microbiology and Immunology, Eulji University School of Medicine, 2Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

The complement system is an important component of immune defense against pathogens. Physiologically, complement system can induce inflammation, opsonization, and MAC-mediated cell lysis. In addition to the roles in a first defense system, complement participates in diverse immunological processes including control of adaptive immunity, removal of apoptotic cells, angio-genesis, etc. Inflammation is one of pathologic features of Kaposi's sarcoma but little has been known for the activation of complement system in KSHV. We have reported complement activation on latent KSHV-infected cells, providing survival signals for the KSHV-infected cells. Here, we aimed to investigate whether complement system is activated during acute KSHV-infection in HUVECs. Despite several complement regulatory proteins were highly up-regulated after KSHV infection, treatment of human serum induced the depositions MAC on the KSHV-infected cells, which would be triggered by binding of properdin on the cell surface. Meanwhile, the exosome released from KSHV-infected cells induced the binding of properdin on the surface of KSHV-infected and naïve endothelial cells. Furthermore, the activated complement system supported the survival of KSHV-infected cells during lytic replication. Our findings suggest a novel mechanism of complement activation in acute KSHV infected HUVECs and provide a further insight on the microenvironment during KSHV-infection in human endothelial cells. [Supported by grants from NRF.]

D040

Significant Differences between Tick Bite Sites and Mite Bite Sites on Humans

Choon-Mee Kim1, Na-Ra Yoon2, and Dong-Min Kim2*1Premedical Science, College of Medicine, Chosun University, 2Department of Internal Medicine, College of Medicine, Chosun University

Identification of mite and tick bite sites provides an important clinical clue for diagnosis. To date, there has been no comparative study on preferred bite sites between mites and ticks in patients. This study was conducted by reviewing medical records and through a field study. For mite bite sites, eschars were checked on 506 patients with scrub typhus, as confirmed using immunofluorescence assay or nested polymerase chain reaction targeting the 56-kDa gene. Tick bite sites were identified and marked on a diagram for 91 patients who experienced tick bites within the previous year through a field epidemiological investigation. The mite bite and tick bite sites were compared. The most frequently observed mite bite sites were the anterior chest, including the axillary region (29.1%), and abdominal region, including the inguinal area (26.1%). Tick bite sites were most frequently found on the lower extremities (33.0%), followed by the abdominal region, including the inguinal area (26.4%), and upper extremities (26.4%). The distribution was significantly different between mite bite and tick bite sites (p<0.001). There was a statistically significant difference in the mite bite sites (p=0.001) but not the tick bite sites (p=0.985) between men and women.To our knowledge, this is the first report of the differences between tick bite sites and mite bite sites, which is intended to help clinicians with rapid diagnoses of mite or tick borne infections.

D041

Utility of Tick-bite Site Samples for the Diagnosis of Human Granulocytic Anaplasmosis

Choon-Mee Kim1 and Dong-Min Kim2*1Premedical Science, College of Medicine, Chosun University, 2Department of Internal Medicine, College of Medicine, Chosun University

Human granulocytic anaplasmosis (HGA) is a tick-borne infectious disease caused by Anaplasma phagocytophilum, an obligate intracellular bacterium. Until now, the utility of tick-bite site samples for HGA diagnosis has not been reported. Using a patient’s buffy coat and tick-bite site crust, we performed polymerase chain reaction (PCR) testing using Ehrlichia or Anaplasma-specific primers. PCR with Anaplasma-specific primers and buffy coat and crust specimens obtained before doxycycline administration was positive, whereas PCR with buffy coat specimens obtained six days after doxycycline administration was negative. However, PCR with tick-bite site crust specimens obtained six days after doxycycline administration remained positive. Therefore, like buffy coat PCR, PCR with a tick-bite site crust appeared useful in the early diagnosis of HGA. This is the first study to suggest that tick-bite site samples are useful for HGA diagnosis in patients who have already been treated with antibiotics such as doxycycline.

Poster

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D042

Clinical Usefulness of Conventional PCR Targeting the 16S Ribosomal RNA for the Diagnosis of Scrub Typhus

Choon-Mee Kim1 and Dong-Min Kim2*1Premedical Science, College of Medicine, Chosun University, 2Department of Internal Medicine, College of Medicine, Chosun University

Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi. Delays in diagnosis and antibiotic administration can cause fatal complications. Therefore, a rapid and early diagnosis is critical.We retrospectively evaluated the accuracy of conventional PCR targeting the 16S rRNA gene (16S C-PCR) using the Ot-16sRF1/Ot-16sRR1 primers for the diagnosis of scrub typhus. We examined blood specimens from 115 adult patients confirmed to have scrub typhus, 52 patients confirmed to have characterized illness and 35 febrile patients who had neither scrub typhus nor any characterized illness. To evaluate the clinical usefulness of this new method, we compared its diagnostic effectiveness with that of the following methods: C-PCR targeting the 47-kDa, 56-kDa, and groEL genes; nested PCR targeting the 47-kDa and 56-kDa genes; and real-time PCR targeting the 47-kDa gene.The C-PCR with the Ot-16sRF1/Ot-16sRR1 primers detected O. tsutsugamushi infection with a sensitivity of 87% and a specificity of 100%. Other C-PCR amplified the 47-kDa, 56-kDa, and groEL genes with lower sensitivities of 3%, 8%, and 66%, respectively. Nested PCR amplification of the 47-kDa and 56-kDa genes showed sensitivities of 81% and 80%, respectively, whereas real-time PCR of the 47-kDa gene exhibited a sensitivity of 76%. The 16S C-PCR using the Ot-16sRF1/Ot-16sRR1 primers is simple, clinically useful.

D043

Host Transcriptional Profiles of Acinetobacter baumannii-Infected Human Lung Cancer Cell

Sang-Yeop Lee1, Edmond Changkyun Park1,2, Sung Ho Yun1, Chi Won Choi1, Hayoung Lee1, Gun-Hwa Kim1,3, and Seung Il Kim1*1Division of Bioconvergence Analysis, Korea Basic Science Institute, 2Department of Bio-Analytical Science, University of Science and Technology, 3Department of Functional Genomics, University of Science and Technology

Multidrug resistant Acinetobacter baumannii is a predominant causative organism of pneumonia by hospital outbreaks. However, details of infection mechanism of A. baumannii were not elucidated well. To investigate host response by A. baumannii infection, A549 lung cancer cells were treated with whole bacterial cells, membrane fraction, or outer membrane vesicles of A. baumannii and transcriptomic signature of the A549 cells were analyzed. As a result, whole cell-infected A549 cells were differentially expressed 2,324 genes compared to untreated control A549 cells. Most of those genes were related to biological process of cell cycle, apoptosis and cell death. Among them, Jun, GADD45A and IL8 were highly expressed in A. baumannii-infected A549 cells. Jun and GADD45A were commonly involved in mapk signaling pathway. In A. baumannii membrane factions-treated A549 cells, 1,039 genes were differentially expressed comparing to untreated control A549 cells. Those genes were mainly participated in the process of cell cycle arrest and inflammation response. In A549 cells treated with A. baumannii outer membrane vesicles, expression of 1,257 genes were changed and those genes were related to apoptosis and cell death. These results indicate that host response of outer membrane vesicle-treated A549 cells were more similar to whole cell-infected A549 cells than membrane fraction-treated A549 cells.

D044

Conditionally Pathogenic Gut Microbes Promote Larval Growth by Increasing Redox-Dependent Fat Storage in High Sugar Diet-Fed Drosophila melanogaster

Tae Woong Whon, Na-Ri Shin, Mi-Ja Jung, Dong-Wook Hyun, Hyun Sik Kim, Pil Soo Kim, and Jin-Woo Bae*


Changes in the composition of the gut microbiota contribute to the development of obesity and subsequent complications associated with metabolic syndrome. However, the role of increased numbers of certain bacterial species during the progress of obesity remains unclear. Here, we show that chronic feeding of a high sugar diet (HSD) to Drosophila melanogaster resulted in a predominance of resident uracil-secreting bacteria in the gut. Axenic insects mono-associated with uracil-secreting bacteria or supplemented with uracil under HSD conditions promoted larval development. Redox signaling induced by bacterial uracil promoted larval growth by regulating sugar and lipid metabolism via activation of p38a mitogen-activated protein kinase. The present study identified a new redox-dependent mechanism by which uracil-secreting bacteria (previously regarded as opportunistic pathobionts) protect the host from metabolic perturbation. Thus, changes in the gut microbiota play an important role in alleviating deleterious diet-derived effects such as hyperglycemia.[Supported by a grant from the Mid-career Researcher Program (2011- 0028854 to J.-W.B.) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP), and from the Basic Science Research Program (2015019390 to T.W.W.) through the NRF funded by the Ministry of Education.]

D045

Phylogenetic Analysis of Leptospira spp. in Wild Rodent at Gwangju Metropolitan Area, Republic of Korea 2014~2015

Jung Wook Park1, Sun Hee Kim1, Duck Woong Park1, Hye Jung Park1, So Hyang Jeong1, Mi Hee Seo1, Yong Seok Lee1, Jae Keun Chung1, Hyun Jae Song2, and Jung Yoon Lee2, and Dong Min Kim3*1Health and Environment Institute of Gwangju, 2Gwangju Health University3Chosun University, Medical College

Leptospirosis is an important zoonotic disease worldwide, acquired through direct contact with animal reservoirs and an environment contaminated by their urine. To obtain a better understanding of prevalence in wild rodents at Gwangju metropolitan area, we conduct a survey from September, 2014 to December, 2015.Wild rodents were monthly captured by sherman live trap. Kidney specimens of 238 wild rodents from 2 area were examined by PCR based 3 primer (flaB, secY, 16srRNA). Leptospiral DNA was amplified in 21.8% (52/238) including 5.9% (14/238) in flaB primer, 8.8% (21/238) in secY primer and 7.1% (17/238) in 16S rRNA. Of them, 17 sequences (32.7%; 17/52) were analysed. They had a homology with Leptospira spp. of GenBank.Our results provides a baseline data for Leptospirosis in Gwangju metropolitan area. To predict reemerging of this disease in human, needs continuous wildlife monitoring [Suppoted by Healh & Environment Research institute of Gwangju city.]


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D046

Aggregatibacter actinomycetemcomitans Lipopolysaccharide- mediated Induction of Chemokines MCP-1, MIP-1α, and IP-10 Occurs via Distinct Intracellular Signaling Pathways in Murine Macrophages

Ok-Jin Park, Min-Kyung Cho, Cheol-Heui Yun, and Seung Hyun Han*

Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium frequently isolated from lesions of patients with localized aggressive periodontitis. Lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria, stimulates innate immune cells via Toll-like receptor 4 (TLR4) to initiate inflammatory responses. In this study, we purified LPS from A. actinomycetemcomitans (AaLPS) and investigated its ability to induce the expression of chemokines. AaLPS induced the expression of chemokines, MCP-1, MIP-1α, and IP-10, leading to the infiltration of peripheral blood mononuclear cells in a transwell system. Although TLR4 was essential for the induction of all these chemokines by AaLPS, MCP-1 and MIP-1α expressions were MyD88-dependent, but IP-10 expression was MyD88-independent. Furthermore, the activation of ERK and JNK were necessary for the expression of MCP-1 and MIP-1α, whereas p38 MAP kinase and JNK activations were required for IP-10 expression. In addition, IFN-β/STAT1 signaling was exclusively involved in IP-10 expression but not in MCP-1 or MIP-1α expression. AaLPS also activated NF-κB, AP-1, NF-IL6, and ISRE, all of which are involved in chemokine gene expression. These results suggest that AaLPS induces the expression of chemokines MCP-1, MIP-1α, and IP-10 through TLR4. Further, the induction of MCP-1 and MIP-1α requires MyD88, ERK, and JNK, whereas the induction of IP-10 requires JNK, p38 MAP kinase, and IFN-β/STAT1.

D047

Acquisition of Chemoresistance and Other Malignancy-related Features of Colorectal Cancer Cells are Incremented by Ribotoxic Mycotoxin and Antibiotics

Chang-Kyu Oh1, Dongwook Kim2, Seung-Joon Lee3, Seong-Hwan Park3, and Yuseok Moon3*1Department of Biomedical Sciences, Pusan National University School of Medicine2National Institute of Animal Science, RDA3Department of Biomedical Sciences, Pusan National University

Colorectal cancer (CRC) as an environmental disease is largely influenced by accumulated epithelial stress from diverse environmental causes. We are exposed to ribosome-related insults including ribosome-inactivating stress (RIS) from mycotoxins and antibiotics, but their physiological impacts on the chemotherapy of CRC are not yet understood. Here, we revealed the effects of RIS on chemosensitivity and other malignancy-related properties of CRC cells. First, RIS led to bidirectional inhibition of p53-macrophage inhibitory cytokine 1 (MIC-1)-mediated death responses in response to anticancer drugs by either enhancing ATF3-linked antiapoptotic signaling or intrinsically inhibiting MIC-1 and p53 expressions, regardless of ATF3. Second, RIS enhanced epithelial mesenchymal transition (EMT) and biogenesis of cancer stem-like cells (CSC) in an ATF3-dependent manner. These findings indicated that gastrointestinal exposure to RIS interferes with the efficacy of chemotherapeutics, mechanistically implicating that ATF3-linked malignancy and chemoresistance can be novel therapeutic targets for treatment of environmentally aggravated cancers. [This work was carried out with the support of the Cooperative Research Program for Agriculture, Science & Technology Development (Project No. PJ01093206)" Rural Development Administration, Republic of Korea).]

D048

Acute Gastroenteritis Surveillance from Diarrheal Patients in Gwangju, Korea, During 2015

Seon Kyeong Kim, Hye-young Kee, Tae Sun Kim, Eun-hye Jo, Ji Hyun Shin, Dong Ryong Ha, Eun-Sun Kim, and Kye Won Seo*

Health & Environment Institute of Gwangju

Acute gastroenteritis (AGE) has been recognized as one of the most common diseases in humans and continues to be a major public health problem worldwide, primarily occurs in infants and young children in both and developed developing countries. A total of 1,525 samples of rectal swabs and stool specimens of diarrheal patients were collected from 9 pediatric hospitals and internal medicine during 2015. For detection of 10 species bacterial pathogens including salmonella and 5 enteric virus including norovirus, We analyzed by the isolation culture, biochemical identification and enzyme immunoassay, reverse transcription-PCR, DNA sequencing. Enteric viruses and bacteria were detected in 447 (29.3%) and 393 (25.8%) specimens, respectively. Among the enteric viruses detected, norovirus 305 (20.0%) and rotavirus 88 (5.8%) were the predominant causative agents, followed by astrovirus 28 (1.8%), adenovirus 19 (1.3%) and sapovirus 7 (0.5%). Among the pathogenic bacteria, Salmonella accounting for 115 (7.5%) was the most frequent pathogen, followed by Staphylococcus aureus accounting for 108 (7.1%), pathogenic E. coli accounting for 88 (5.7%). The epidemic peaks of the enteric viruses were October to January for norovirus, January to April for rotavirus. The seasonal activity of rotavirus was shifted from winter to late spring. Sequencing of the norovirus strains revealed that GII.4/2012 (80/305, 26.2%) sydney variant was the most common genotype and followed by GII.3 (35/305, 11.5%) and GII.17 Kawasaki (28/305, 9.2%) strains. Ongoing surveillance will help further elucidate trends, identify gaps, and assess the effects of future interventions on reducing epidemic gastroenteritis.

D049

ɩNKT Cell Sensitization during Neonatal Respiratory Syncytial Virus Infection Induces Severe Pulmonary Pathology in Re-infected Adult Mice

Seung Young Lee1, Youran Noh1, Semi Rho1, Jung Hyun Goo1, Min Jung Kim1, Chang-Yuil Kang2, Man Ki Song1, and Jae-Ouk Kim1*1Laboratory Science, International Vaccine Institute2College of Pharmacy, Seoul National University

Respiratory syncytial virus (RSV) is a major viral pathogen that causes severe lower respiratory tract infections in human infants worldwide. Infants with severe RSV bronchiolitis tend to experience more wheezing and asthma in later childhood. Because invariant natural killer T (ιNKT) cells are associated with the pathology of asthma, we investigated whether neonatal ιNKT cells are involved in aggravation of pulmonary diseases following RSV infection. Intranasal exposure of the ιNKT cell ligand, α-galactosylceramide (α-GC), with RSV primary infection in neonatal mice elicited neither cytokine production nor pulmonary eosinophilia despite of the presence of both CD1d+ cells and ιNKT cells. Interestingly, in adult mice re-infected with RSV, neonatal ιNKT cell sensitization by α-GC during RSV primary infection resulted in much higher levels of pulmonary Th2 cytokines and elevated eosinophilia with severe airway hyperresponsiveness. In contrast, α-GC priming of adults during RSV re-infection did not induce more severe airway symptoms than RSV re-infection in the absence of α-GC. α-GC co-administration during RSV primary infection facilitates RSV clearance regardless of age, but the viral clearance following re-infection was not ιNKT cell-dependent. This study suggests that neonatal ιNKT cell sensitization during RSV primary infection is strongly associated with exacerbation of pulmonary diseases following RSV re-infections in adulthood.[Supported by KNRF (NRF-2014R1A1A3049897)]

Poster

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D050

B Cell Infection by Kaposi’s Sarcoma-associated Herpesvirus

Jinjong Myoung

Korea Zoonosis Research Institute, Chonbuk National University

Kaposi’s sarcoma-associated herpesvirus is known to infect B cells and endothelial cells. Among them, B cells seem to serve as the main virus reservoir and migration vector in Kaposi’s sarcoma. KSHV infection in B cells induces two very different B cell lymphomas: multicentric Castleman’s disease (MCD) and primary effusion lymphoma (PEL). However, interestingly, peripheral blood B cells have been resistant to KSHV infection ex vivo, which deters detailed delineation of tumorigenesis mechanisms in B cells. Here, we report that human lymphoid aggregate cultures (HLAC’s), established from tonsils, are susceptible to KSHV infection. Infected B cells display spontaneous lytic replication, thus more closely resembling the pathobiology of MCD. This in vitro model will undoubtedly provide a useful model for studying KSHV biology in B cells.

D051

OX40 and 4-1BB Differentially Inhibit KSHV Replication in B Cells and Endothelial Cells

Jinjong Myoung


OX40 belongs to the tumor necrosis factor superfamily, and is known to modulate T cell activation and memory T cell generation. Previously, Byun et al elegantly demonstrated genetic deficiency of OX40 leads to childhood Kaposi’s sarcoma, underscoring the importance of OX40 co-stimulation in the generation of KSHV-specific T cell activation. Here, we report for the first time that ligation of OX40L on endothelial cells with recombinant OX40 effectively inhibits KSHV replication. Interestingly, previously unsuspected roles of 4-1BB in KSHV replication were highlighted in infected endothelial cells as well. On the contrary, in infected B cells, neutralization of 4-1BB- 4-1BBL, but not that of OX40-OX40L, inhibits KSHV replication, indicating differential role of OX40 in two different main targets of KSHV in vivo.

D052

Human Fucosyltransferase 2 Expression in Mouse

Jinjong Myoung


Human norovirus (hNoV) accounts for over 90% of virus-mediated acute gastroenteritis, thus posing a serious threat to public health. It was first identified in 1968, however, due to the lack of susceptible cell lines and animal model, progress in its pathophysiology has been serious hindered. As human fucosyltransferase 2 (hFUT2) seems to be the disease determinant, we have set out to develop hFUT2-transgenic mice as a small animal model for hNoV. hFUT2-expressing transgenic mice were backcrossed to C57BL/6 and BALB/c background. When hFUT2-Tg with B6 background were inoculated with hNoV (1 × 106 genome copies/mouse), low levels of viral replication was detected in various tissues, including liver, small intestines, and spleen. Unfortunately, as backcrossing increases hFUT2-Tg susceptibility to hNoV decreases, highlighting the importance of genetic background for viral susceptibility. Further improvement needs developing for the generation of suitable small animal models.

D053

KSHV Infection in Established B Cells Require Cell-associated Viruses

Jinjong Myoung


Kaposi’s sarcoma-associated herpesviral infection in primary B cells provides an exciting model for the investigation of KSHV biology in B cells. Ex vivo infection of tonsillar B cells display a moderate level of survival advantage over uninfected B cells. However, infected tonsillar B cells are neither immortalized or transformed, which poses barriers to study biochemical events in B cells upon KSHV infection. As such, many attempts had been made to infect established lymphoblastoid cells by KSHV with no avail. Here, we report immortalized or transformed B cells are rendered susceptible to KSHV infection only when in direct contact with cell-associated viruses. Upon infection, B cells are mostly latently infected, which may provide a model for investigating KSHV-mediated interventions of signal transduction pathways in B cells.


46 | www.msk.or.kr

D054

Jak-STAT Pathway Enhances KSHV Replication in Endothelial Cells

Jinjong Myoung


Since KSHV discovery in the early 1990’s, it has been known that IL-6 is the tumor growth factor in KSHV-mediated malignancies. As IL-6 is the canonical activator of Jak-STAT3 pathway and STAT3 is an oncogene, it has been assumed that IL-6 and Jak/STAT3 are involved in tumorigenesis, but not in viral replication. Here, we report that STAT3 is both necessary and required for efficient viral replication in endothelial cells. As in Kaposi’s sarcoma and multicentric Castleman’s disease viral replication is required for tumor growth, we propose that STAT3-mediated enhancement of KSHV replication may be an important mechanisms for KSHV-mediated tumorigenesis.

D055

Calcineurin Targets Involved in Stress Survival and Fungal Virulence

Hee-Soo Park1,2*, Joseph Heitman2, and Maria E. Cardenas2

1School of Food Science and Biotechnology, Kyungpook National University, 2Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA

Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic TiO2 enrichment and quantitative mass spectrometry. The identified targets include the zinc finger transcription factor Crz1 and proteins whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a bona fide calcineurin substrate, and localization and transcriptional activity of Crz1 are controlled by calcineurin. Several of the calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, and are required for survival at high temperature and for virulence. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings propose that calcineurin controls thermal stress and virulence at the transcriptional level via Crz1 and post-transcriptionally by regulating target factors involved in mRNA metabolism.

D056

Prooxidant Activity of Pyrogallol on Vibrio vulnificus Infection

Ju Young Lim and Young Ran Kim*

College of Pharmacy, Chonnam National University

Vibrio vulnificus, the causative agent of fatal septicemia and necrotic wound infection, is a good model organism for bacterial septicemia studies. We found that the polyphenol pyrogallol protected HeLa cells from repeats- in-toxin A1 (RtxA1)-mediated morphological damage and V. vulnificus-induced cytotoxicity. Pyrogallol also inhibited the growth of V. vulnificus, and the inhibitory effect was more significant in the log phase than in the stationary phase. To investigate the inhibitory mechanism, pyrogallol-induced toxicity was compared between a V. vulnificus catalase–peroxidase (katG−) mutant and the wild-type MO6-24/O strain. The katG− mutant did not show any growth in the presence of pyrogallol (50 μg/ml) until 24 h, while the wild-type strain showed growth recovery in the stationary phase. The pathogen growth inhibition by pyrogallol was reduced by catalase treatment. These results indicate that the mechanism by which pyrogallol inhibits the growth of V. vulnificus may involve polyphenol-induced prooxidant damage.

Keywords: pyrogallol, prooxidant activity, catalase, Vibrio vulnificus, katG mutant

D057

Role of Host Cell Filamin A in Vibrio vulnificus RtxA1 Toxin-induced Cytoskeletal Rearrangement and Cytotoxicity

Ju Young Lim and Young Ran Kim*

College of Pharmacy, Chonnam National University

Cytoskeletal rearrangement and acute cytotoxicity are observed in Vibrio vulnificus RtxA1 intoxication. Amino acids 1491-1971 of V. vulnificus 29307 RtxA1 toxin exhibits approximately 25% identity with ezrin, radixin, moesin (ERM) family protein sequences that function as linkers between the plasma membrane and actin cytoskeleton. HeLa cells expressing RtxA1 amino acids 1491-1971 fused to green fluorescence protein (GFP-RtxA1ERM) became rounded and the fusion protein colocalized with actin. Through a yeast two-hybrid screening and subsequent validation by immunoprecipitation assay, we confirmed a specific binding of RtxA1 with host cell filamin A, an actin-crosslinking scaffold protein. Interestingly, HeLa cells intoxicated with RtxA1 appeared to selectively dump out cytoskeleton proteins, such as filamin, actin, and tubulin, to the culture supernatant. Downregulation of filamin A by siRNAs decreased the cytotoxicity of RtxA1 to HeLa cells. Pretreatment with a filamin A-specific antibody decreased the cytotoxicity of V. vulnificus RtxA1. Phosphorylation of JNK and p38 mitogen-activated protein kinases was located below the RtxA1-filamin A binding during the RtxA1-mediated death of HeLa cells. These results suggest that remodeling of cytoskeleton after filamin A interaction with RtxA1 toxin would be an important cellular reaction during the programmed necrotic death of RtxA1 intoxicated cells.

Poster

www.msk.or.kr | 47

E001

Purification, Crystallization, and X-ray Crystallographic Studies on Bacillus stearothermophilus Molybdenum Cofactor-dependent YiiM

Byeol Nam-gung and Sung-il Yoon*

Department of Biomedical Convergence, College of Biomedical Science, Kangwon National University

YiiM is found in a variety of bacteria and plays a major role in the removal of toxic N-hydroxylated base analogues. YiiM converts mutatgenic 6-N- hydroxylaminopurine (HAP) to adenine in a molybdenum cofactor (MoCo) dependent manner. YiiM belongs to the MoCo sulphurase C-terminal domain (MOSC) protein superfamily. The cofactor-binding mode and enzymatic mechanism of the MOSC family remain to be revealed. As a first step to define the biochemical activity and structural features of YiiM, we have performed expression, purification and X-ray crystallographic studies on Bacillus stearothermophilus YiiM (bsYiiM). bsYiiM was overexpressed in an Escherichia coli expression system and purified to homogeneity by chromatographic methods. bsYiiM crystals were obtained in PEG conditions and diffracted X-ray to 2.0 Å resolution. The crystals belonged to space group P212121 with one molecule in the asymmetric unit. The bsYiiM structure was determined by molecular replacement and is currently under model building and refinement. The final structure would allow us to predict the MoCo-binding site of MOSC proteins and provide their catalytic mechanism.

E002

Anthranilate Deteriorates Bacterial Biofilms

Xi-Hui Li1,2,3, Soo-Kyung Kim1,2,3, Jungmin Oh1,2,3, and Joon-Hee Lee1,2,3*1Lab of Microbiology, 2College of Pharmacy, 3Department of Pharmacy, Pusan National University

Anthranilate is an important intermediate for the syntheses of tryptophan and Pseudomonas quinolone signal in Pseudomonas spp. While some bacteria, including P. aeruginosa, degrade tryptophan to anthranilate through a kynurenine pathway using the kynBAU genes, other bacteria harboring tnaA gene encoding tryptophanase that converts tryptophan into indole, pyruvate, and ammonia produce indole from tryptophan degradation (for examples, Escherichia coli, Vibrio vulnificus). Therefore, bacterial community that exists in tryptophan-rich environments contains anthranilate or indole (or both) necessarily. It has been recently reported that anthranilate deteriorates the biofilm structure by reducing intracellular c-di-GMP levels and modulating the expression of Psl, Pel, and alginate in P. aeruginosa. We investigated the anthranilate effect on biofilm formation of various bacteria and the underlying mechanism of the anthranilate effect.Static and flow-cell systems were used for biofilm formation and direct confocal microscopic observation was carried out to monitor the anthranilate effect on biofilm formation. We also measured bacterial motility and intracellular c-di-GMP levels.Anthranilate has a significant deteriorating effect on biofilms of Vibrio vulnificus, Bacillus subtilis, and Staphylococcus aureus. And anthranilate significantly enhanced swimming and swarming motility of V. vulnificus and B. subtilis.[Supported by grants from KRF]

E003

Ornithine Lipid-mediated Regulation of Virulence and Biofilm Formation in Pseudomonas aeruginosa

Soo-Kyung Kim1,2,3, Xi-Hui Li1,2,3, and Joone-Hee Lee1,2,3*1Lab of Microbiology, 2College of Pharmacy, 3Department of Pharmacy, Pusan National University

Ornithine lipids (OLs) are bacteria-specific lipids that are widely found in outer membrane of many Gram (-) bacteria, but not detected in Eukarya and Archaea. Pseudomonas aeruginosa produces OL under phosphate-limiting conditions and has olsBA operon encoding acyltransferases that functions the OL biosynthesis. This olsBA operon is highly expressed under phosphate- limiting condition. OlsB has similar structure with LasI, an acyl-homoserine lactone synthase. We addressed how OLs modulate the biofilm formation and virulence of P. aeruginosa during the infection to host cells. The virulence of P. aeruginosa was investigated by using two host models. The overproduction of OLs alleviated the virulence of P. aeruginosa by reducing the quorum sensing activity. We suggest that the olsBA overexpression sequesters the cellular acyl-group pool toward the OL synthesis from acyl homoserine lactone synthesis. The OL effect on biofilm formation was analyzed in flow cell system. OLs enhanced the biofilm formation of P. aeruginosa. Since the hydrophobicity of cell surface increased by the OL overproduction, this may facilitate the attachment of cells to surface. The host response to OLs was investigated by measuring the expression of inflammatory factors in mouse macrophage cells. OLs reduced the production of inflammatory factors such as iNOS, COX-2, PEG2, and nitric oxide in host cells, suggesting that OL can modulate the host immune response.[Supported by grants from KRF]

E004

Dissection of the HOG Pathway Activated by Hydrogen Peroxide in Saccharomyces cerevisiae

Young Mi Lee1,2, Eun Jung Kim1,2, Ji Eun An3, Ye Ji Lee4, Eun Yong Choi4, Won Ja Choi2,3,4, Eun Pyo Moon5, and Wan Kee Kim1*1Department of Pharmacology, School of Medicine, Ajou University, 2Division of Ecological Sciences, College of Natural Sciences, 3Department of Life Sciences and Division of Ecological Sciences, College of Natural Sciences, 4Interdisciplinary Program of EcoCreative, College of Natural Sciences, Ewha Womans University, 5Department of Life Sciences, College of Natural Sciences, Ajou University Cells usually cope with oxidative stress by activating signal transduction pathways. In the budding yeast Sacchromyces cerevisiae, the high osmolarity glycerol (HOG) pathway has been implicated in transducing the oxidative stress-induced signal, but the underlying mechanisms are not well defined. Based on phosphorylation of the mitogen-activated protein kinase (MAPK) Hog1, we revealed that the signal from hydrogen peroxide (H2O2) flows through Ssk1, the response regulator of the two-component system of the HOG pathway. Downstream signal transduction into the HOG MAPK cascade requires the MAP kinase kinase kinase (MAPKKK) Ssk2 but not its paralog Ssk2. Activation of Ssk2 occurs in both Ssk1-dependent and -independent manners, culminating in Hog1 phosphorylation via the MAPKK Pbs2. H2O2- activated Hog1 is retained in the cytoplasm, but is still able to activate the cAMP- or stress-responsive elements by unknown mechanisms. This signaling pathway may be used to identify oxidative stress-related MAPKKKs of other species including mammals based on the ability to complement the SSK2 deletion mutant and subsequently activate Hog1 in response to H2O2.


48 | www.msk.or.kr

E005

Overexpression of OLE1 Enhances Stress Tolerance and Constitutively Activates the MAPK Hog1 through the MAPKKK Ssk2

Ye Ji Lee1, Olviyani Nasutution2, Young Mi Lee3, Eun Jung Kim3, Wan Kee Kim3, and Won Ja Choi1*1Interdisciplinary Program of EcoCreative, 2Division of Life and Pharmaceutical Sciences, Ewha Womans University, 3Department of Pharmacology, School of Medicine, Ajou University

Previously, it was found that the mRNA level of OLE1 increases significantly in Saccharomyces cerevisiae cells exposed to acetic acid. Compared with control, an OLE1-overexpressing strain displayed better proton efflux, lower membrane permeability and lessened internal hydrogen peroxide content, and enhanced tolerance to various stresses. The enhanced stress tolerance was considerably diminished when OLE1 was overexpressed in a strain deleted for HOG1, which encodes the mitogen-activated protein kinase (MAPK) Hog1 of the high osmolarity glycerol (HOG) pathway, demonstrating its Hog1 dependency. We further found that OLE1 overexpression constitutively activates Hog1, which remains in the cytoplasm. This Hog1 activation was accomplished through the MAPK kinase kinase (MAPKKK) Ssk2, but not through Ste11 and Ssk22, the other MAPKKKs of the HOG pathway. Despite its cytoplasmic location, activated Hog1 was able to activate the expression of its canonical targets such as CTT1, HSP12, and STL1, and further the cAMP response element and stress response element present in the promoter. On the other hand, OLE1 overexpression neither caused nor relieved the endoplasmic reticulum stress. Individually or in combination, the physiological and molecular changes caused by OLE1 overexpression may contribute at least in part to enhanced tolerance to various types of stress.[Supported by BK21 plus]

E006

Structural and Functional Studies on Uracil DNA Glycosylase from Bradyrhizobium japonicum

Vinod Vikas Patil1,2, Ullas Valiya Chembazhi3, Biak Sang Pau3, Ravi Tiwari4, Umesh Varshney3, and Eui-Jeon Woo1,2*1Korea Research Institute of Bioscience and Biotechnology, 2University of Science and Technology, 3Department of Microbiology & Cell Biology, IISc, Bangalore, India, 4School of Veterinary and Life Sciences, Murdoch University, Western Australia

Stress conditions during plant rhizobia interactions induces DNA damage in rhizobia. The deamination of cytosine to uracil in DNA is one of the common cause for DNA damage that affect overall fitness of bacteria. Uracil DNA Glycosylase is an enzyme that repair DNA by base excision pathway. Here we report a novel protein Q89XRO_BRAJA (BjUng) isolated from Bradyrhizobium japonicum, an organism in which no classical homologue of family-1 UDG have been identified. Kinetic and biochemical characterization of BjUng reveals it as a single strand specific UDG, which is also active on 5-hydroxymethyl uracil and xanthine. Unlike family 1 UDGs, BjUng is impervious to inhibition by Ugi, a phage protein which specifically inhibits the family 1 UDGs, and the abasic DNA (AP-DNA). BjUng homologues are present in many other microbes and our phylogenetic analysis reveals that this protein constitutes a previously unidentified family of UDGs. Crystal structure of BjUng shows striking similarity with the family 1 UDGs in terms of key active site residues and overall fold, suggesting its origins through divergence. Bjung exist as a dimer with seven α-helix and six β- sheets. One ligand binding site is present per monomer of Bjung. Asp57, Glu62, Leu69, Asn94, His168 are critical residues involved in uracil binding. Our studies on Bjung shed light on structural and functional aspect of DNA repair system in rizobacteria.

E007

Structural Analysis of Ligand Bound Complex of UdgX: A Unique Uracil DNA Glycosylase Superfamily Binding Uracil DNA

Woo-Chan Ahn1,2, Min-Ho Lee1, Pau Biak Sang3, Umesh Varshney3, and Eui-Jeon Woo1,4*1Korea Research Institute of Bioscience and Biotechnology, 2Department of Biological Sciences, KAIST Institute for the Biocentury, Korea Advanced Institute of Science and Technology, 3Department of Microbiology & Cell Biology, IISc, Bangalore, India, 4University of Science and Technology Uracil DNA glycosylases recognize and excise Uracil from DNA as the first step of base excision repair system, an important pathway of DNA repair system. UdgX, the most recently discovered UDG from Mycobacterium smegmatis, specifically recognizes uracil in DNA. It tightly binds to uracil in DNA. The by binding that is even stable in higly reducing conditions in SDS gel and heat treatment with a signature loop, KRRIH. In previous biochemical report (Sang et al (2015) 43(17) Nucl. Acids Res.) mutation of the unique loop, KRRIH, leads UdgX to cleave uracil from DNA as other Udg does. Here, we report the crystal structure of UdgX, UdgX capturing uracil, UdgX H109S mutant, and complex with UdgX and DNA containing uracil. Considering this enzyme belongs to Udg family4, overall structure and catalytic residues of UdgX are well conserved. The structure with uracil in active site pocket also verifies the conservation of catalytic motif among Udgs. In structure of the complex of UdgX and DNA with uracil, however, presents unique features which has not reported before. The structure of deoxyuridine in UdgX-DNA complex shows unstable conformational strain, which imply the catalytic mechanism of Udgs. The unique loop possessing His109 locates very close to 3’ phosphate of deoxyuridine, which suggests the ground for tight binding of UdgX and DNA possessing uracil.

E008

Elucidation of Regulatory Genes for Enhancement of Rapamycin Production

Jin A Jung, Eun ji Kim, Jae-yeon Hwang, Myoun Su Kim, Shi Ying Jin, Na Ryeong Lee, and Yeo Joon Yoon*

Department of Chemistry and Nano Science, Ewha Womans University

Rapamycin is a 31-membered polyketide macrolide produced by Streptomyces rapamycinicus. It possesses various biological and pharmacological activities including antifungal, immunosuppressive, and anti-aging activities. However, the regulatory mechanism of the pathway-specific regulators for rapamycin biosynthesis has only been partially revealed. Sequence analysis of the rapamycin biosynthetic gene cluster showed that three putative negative regulatory genes rapY, rapS, and rapR indicated high sequence similarity to the known TetR family proteins, response regulator, and histidine kinases, respectively. Overexpression of each three genes significantly reduced rapamycin production, while deletion of rapY and rapS induced increased level of rapamycin production by 4.6-fold and 3.7-fold, respectively, compared to that of the wild-type strain. Gene expression analysis by RT-PCR showed that rapS negatively controlled the expression of rapY and rapX encoding a putative ABC-transporter was negatively controlled by rapY. Interestingly, overexpression of rapX in the rapS deletion mutant accomplished an increase in rapamycin production compared to the wild-type strain. These results demonstrate the roles of rapY, rapR, and rapS as negative regulators of rapamycin biosynthesis in S. rapamycinicus and shed light on understanding the complex regulatory mechanism and increasing the level of rapamycin production in industrial strains. [Supported by NRF, ICT and MISP.]

Poster

www.msk.or.kr | 49

E009

Localization of an Acid Responsive Element in the Laccase Promotor Expressed at Low pH in Coprinellus congregatus

Su Yeon Kim1, Linh Trieu Dieu Nguyen1,2, and Hyung Tae Choi1*1Molecular Microbiology Lab, Department of Biochemistry, Kangwon National University, 2Vietnam

Coprinellus congregates, an inky cap, synthesizes and secretes laccase at acidic pH (pH 4.0-4.5) transiently only in the dikaryotic mycelium. Different lengths of expression vectors were constructed using the vector pBARGEM7-1 inserted with green fluorescent protein (GFP) reporter gene regulated by different lengths of the laccase promoter (F0-F5) obtained from 5’-sequential deletion constructs, and they were introduced to wild type strains (a1 & a2) to determine the dikaryon-responsive and acid-stress responsive DNA elements. The transformants were confirmed by PCR with the specific primers which were used in cloning. Monokaryotic transformants of C.congregatus monokaryons were used to generate dikaryons. These transformants were grown in neutral pH liquid media, and then transferred to acidic media to examine the GFP expression by Confocal Microscope. The homozygotic transformants which had full-length (2.0 kb promoter) showed the GFP expression under acidic condition, while the shortest- length-promoter did not show GFP expression. We derived a conclusion that acid stress element localize in the various promotor region between cloned length (F0-F5). We have also constructed shorter expression vector which had shorter lengths of promoter (F6, F7) to confirm this experiment.[Supported by grants from University-Industry Cooperation Foundation]

E010

The Sugar-dependent Regulation of C-di-GMP and Signal Transduction System

Kyoo Heo1, Young-Ha Park1, and Yeong-Jae Seok1,2*1Department of Biological Sciences and Institute of Microbiology, 2Department of Biophysics and Chemical Biology, Seoul National University

Vibrio cholerae, the causative pathogen of severe diarrheal disease, inhabits various niches, including human host. Previous studies uncovered that V. cholerae undergoes adaptation to these environments, which alter their behaviors and various virulence gene expressions. However, the exact mechanism regulating these adaptations has yet to be found. Recent studies have suggested that c-di-GMP, a common secondary messenger found in most bacteria, is involved in the regulation of the virulence of many pathogens, including V. cholerae, which is dependent on various factors such as biofilm formation, motility and toxin production. There are more than 50 proteins involved in controlling the concentration of c-di-GMP in Vibrio cholerae. Diguanylate cyclases which have a GGDEF domain synthesize c-di-GMP from two molecules of GTP, while diguanylate phosphodiesterases containing an EAL or HD-GYP domain have c-di-GMP hydrolase activity. But it is poorly understood which signals regulate activities of these proteins. Here, our study has found that an EAL domain-containing protein interacted with EIIAGlc, a component of the glucose-specific phosphotransferase system (PTS). EIIAGlc regulates the c-di-GMP hydrolase activity of this protein depending on its phosphorylation state. These results suggest that V. cholerae could modulate the level of c-di-GMP in response to sugar sources through PTS component.[Supported by the SamsungScience and TechnologyFoundation under SSTF-BA1501-13]

E011

Trans-4-hydroxy-l-proline: A Novel Constituent of Compatible Solute in Moderate Halophilic Bacteria

Kyung Hyun Kim, Baolei Jia, and Che Ok Jeon*


It has been reported that Halobacillus halophilus, a moderate halophilic bacterium, having a capability to grow a wide range of salinities (0.5–3.0 M NaCl) has a strategy to accumulate osmolytes such as glutamate, glutamine, and proline to cope with osmotic stress under high salt environments. The analysis of organic compounds using 1H-NMR in H. halophilus showed that trans-4-hydroxy-l-proline (Hyp), which has not been reported as a compatible solute in previous studies, increased depending on NaCl concentrations, suggesting that Hyp might be an important compatible solute in H. halophilus. The genome data in GenBank showed that H. halophilus does not harbor a proline 4-hydroxylase (P4H) gene, responsible for the conversion from proline to Hyp. Based on BLAST and Pfam analysis using genes annotated as P4H in other bacteria, a putative P4H gene, which was annotated as a multidrug DMT transporter permease was selected and its expressional enzyme in E.coli strain EL21 showed a clear converting activity from proline to Hyp. Transcriptional analysis also showed that the expression of the putative P4H gene increased at high NaCl conditions. In addition, Hyp was detected from other moderate halophilic bacteria. These results suggest that Hyp may be a ubiquitous compatible solute in moderate halophilic bacteria.[This work was supported by the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ00999302)”, RDA, Republic of Korea.]

E012

Development of a Colorimetric Quantification Method for Characterization of Lactic Acid Bacteria

Min Young Jung, Sung-Oh Sohn, Se Hee Lee, Boyeon Park, Hae Woong Park, and Jong-Hee Lee*

World Institute of Kimchi

Rapid colorimetric methods using various indicator reagents have been developed to monitor bacterial viability. Here, we examined the applicability of a method based on the reduction of resazurin or water-soluble tetrazolium salt-8 (WST-8) to screen lactic acid bacteria (LAB) for tolerance against bile salts and low pH. The resazurin reduction test proved unsuitable for screening LAB such as Lactobacillus plantarum and Leuconostoc mesenteroides since it reacted with lactic acid present in the cultures. LAB growth could be indirectly quantified by measuring WST-8 reduction. This method proved more sensitive than measuring bacterial colony forming units and turbidity at 600 nm. Our results indicate that the WST-8-based method can be used to identify probiotic characteristics in LAB.


50 | www.msk.or.kr

E013

Investigation of the Interaction between HPr and FruR in Vibrio cholera

Chang-Kyu Yoon1, Hey-Min Kim1, Young-Ha Park1, Yeon-Ran Kim1, and Yeong-Jae Seok1,2*1Department of Biological Sciences and Institute of Microbiology, 2Department of Biophysics and Chemical Biology, Seoul National University

Vibrio cholera is known as a human opportunistic pathogen which infects hosts through contaminated water and food. Many bacterial species including V. cholera possess the phosphotransferase system (PTS) as a sugar transport system. The sugar PTS consists of sugar-specific enzyme II proteins and two general components, enzyme I and HPr. These PTS components play critical roles in transport of sugars from outside of cells by sequential phosphoryl transfer. In addition to sugar uptake and phosphorylation, the sugar PTS regulates other functions through protein-protein interaction in a phosphorylation state-dependent manner. Although roles of the PTS have been extensively studied in Escherichia coli, physiological roles of PTS are barely known in V. cholera. In this study, FruR has been found as a new protein partner interacting with HPr in V. cholera. FruR is known as a GalR/LacI superfamily of transcriptional regulator which regulates expression of the genes related in the metabolism and transport of fructose through the direct binding to the cognate operator sequence of the operon of fructose PTS. Only the dephosphorylated form of HPr interacts with FruR and the HPr-FruR complex can bind to DNA fragment containing the putative FruR binding sequence. It is assumed that HPr has an effect on the FruR-dependent regulation as a co-repressor in the presence of glucose.[This work was supported by the Samsung Science & Technology Foundation and National Research Foundation (NRF).]

E014

Generation of a FK506 Analogue through Genetic Engineering of Streptomyces Strain

Xu Zhao, Hea Luying Shin, Heqing Cui, Ji Yoon Beom, Ji Young Lee, and Yeo Joon Yoon*

Department of Chemistry and Nano Science, Ewha Womans University

FK506 is a clinically used immunosuppressant originally isolated from Streptomyces tsukubaensis. In addition to its immunosuppressive action, FK506 has been reported to exhibit antifungal, anti-inflammatory, neuroprotective, and neuroregenerative activities. FK506 is synthesized by a hybrid polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) system. Through our efforts characterizing the functional role of enzymes FkbM and FkbD and identifying the biosynthetic intermediates present in the two parallel pathways for the post-PKS modification of FK506 biosynthesis, a new FK506 analogue (9-deoxo-prolyl-FK506) was isolated and identified from the FkbD deletion mutant of Streptomyces sp. KCTC11604BP. Additionally, 9-deoxo-prolyl-FK506 exhibited reduced in vitro immunosuppressive activity but similar neuroregenerative activity in comparison to FK506. This study demonstrates the successful application of biosynthetic engineering approaches including the deletion of a biosynthetic gene from a gene cluster. In conclusion, this research demonstrates that neuroregenerative drugs can be generated from the FK506 macrolide class of natural products without the side effects attributed to immunosuppression.

E015

OxyR-dependent Gene Expression Involved in Exopolysaccharide Production in Acinetobacter oleivorans DR1

Bora Shin and Woojun Park*

Department of Environmental Science and Ecological Engineering, Korea university

Many bacteria secrete exopolysaccharides (EPS) that contribute to their nutrient trapping, surface attachment, and protection against abiotic or biotic stresses. Like other Acinetobacter species the genome of Acinetobacter oleivorans DR1 contains three distinct EPS operons [two PNAG (poly-N-acetyl glucosamine) operons and the K locus]. Our quantitative RT-PCR analysis showed that the levels of their expression vary under different EPS-producing conditions: H2O2, hexadecane, NaCl, and cold temperature. Morphologically distinct biofilms by those abiotic stresses were observed using confocal laser scanning microscopy, indicating involvement of different cellular components in each biofilm formation. Promoter and expression analyses of EPS genes indicated involvement of the H2O2-sensing OxyR regulator in controlling three EPS operons. Interestingly, OxyR displayed different degree of binding to the promoters of each EPS operon. Biofilm formation was slightly inhibited in the oxyR mutant compared to the wild-type strain under abiotic stress conditions. However, total EPS amounts in the mutant were not changed, which suggested that other factors could contribute to production of different EPS. Further investigation is needed for understanding regulation of OxyR-dependent and -independent EPS productions and their contribution to biofilm formation.[This work was supported by grants from the National Research Foundation of Korea (NRF).]

E016

The Structural Insights of a Cytosol Protein Disulfide Reductase DsbM from Pseudomonas aeruginosa

Inseong Jo1, In-Young Chung2, You-Hee Cho2, and Nam-Chul Ha1*1Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Research Institute for Agricultural and Life Sciences, Seoul National University2Department of Pharmacy, College of Pharmacy, CHA University

In bacteria, many Dsb family proteins play diverse roles in the conversion between the oxidized and the reduced states of cysteine residues of substrate proteins. Most Dsb family proteins target proteins in the periplasmic or extracellular regions of bacteria. Recently, DsbM proteins were found in some Gram-negative bacteria. It is a cytosolic Dsb member with the conserved CXXC motif, and it was implicated in reducing of the cytoplasmic redox sensor protein OxyR in Pseudomonas aeruginosa. Here, we present the crystal structures of DsbM from P. aeruginosa, revealing that it consists of a modified thioredoxin domain containing the CXXC motif and a lid domain surrounding the CXXC motif. In a glutathione-linked structure, a glutathione molecule is linked to the CXXC motif of DsbM and is bound in an elongated canyon-like region in the thioredoxin domain, which is also suited for substrate peptide binding. A striking structural similarity to a human glutathione S-transferase was found as the glutathione binding pocket was shared. We further presented biochemical evidence suggesting that DsbM is directly involved in the reduction of a disulfide of Cys199 and Cys208 in OxyR, resulting in the acceleration of OxyR reduction in the absence of ROS stress. Our findings may help expand the understanding of the diverse roles of Dsb family proteins, which share a common CXXC motif in their cytoplasmic members.

Poster

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E017

Cellular Responses of Hemolytic Bacillus cereus MH-2 Exposed to Epigallocatechin Gallate (EGCG)

Dong-Min Kim, Sang-Kook Park, and Kye-Heon Oh*

Department of Life Science and Technology, Soonchunhyang University

The aim of this work was to investigate the hemolytic activity and characterization of Bacillus cereus MH-2 exposed to EGCG, which produces the hemolytic activity isolated from commercial Ssam-Jang. Initially, the physiological and biochemical characteristics of strain MH-2 was obtained from the Biolog system. Analysis of the carbohydrate utilization profiles based on the GP2 MicroPlate and 16S rRNA sequence also placed MH-2 as a Bacillus cereus with a confidence of 100%, and the strain was designated as B. cereus MH-2, and registered as [KU885976]. Survival of the MH-2 strain with time in the presence of different concentrations of EGCG under sublethal conditions was monitored, and viable counts paralleled the production of the stress shock proteins in this bacterium. Analysis of SDS-PAGE and Western blot using anti-DnaK and anti-GroEL revealed that several stress shock proteins including 70 kDa DnaK and 60 kDa GroEL in strain MH-2 were newly synthesized at different EGCG concentrations in exponentially growing cultures. Scanning electron microscopic analysis demonstrated the presence of protrusions and fused rod forms on the cells treated with EGCG.

E018

Isolation and Cellular Responses of Explosive HMX-degrading Bacterium and Its Morphological Changes Under Sublethal HMX Concentrations

Dong-Min Kim, Sang-Kook Park, and Kye-Heon Oh*


Soil samples were collected from explosive manufacturing plant sites and used for enrichment of microbial consortia with HMX (1,3,5,7-tetranitro- 1,3,5,7-tetrazocane) under aerobic and nitrogen-limiting conditions. Among five isolates from microbial consortia using HMX as substrate for growth, an isolate, HK-5 which has excellent HMX degradability was selected for this work. Both BIOLOG system and 16S rRNA sequencing were conducted to identify the strain, which was assigned to Pseudomonas plecoglossida HK-5. Complete degradation of 75 uM HMX was achieved after 50 days of incubation. Analysis of SDS-PAGE and Western blot revealed that stress shock proteins, DnaK and GroEL, specifically reacting with anti-DnaK and anti-GroEL monoclonal antibodies were produced when cells were treated with HMX. Scanning electron microscopic analysis demonstrated the presence of cells with wrinkled surfaces containing perforations and irregular rod-shaped forms after exposure to HMX.

E019

Physiological Activities of Two Wine Yeasts, Pichia Species Isolated from Crushed Grapes

Sang-Kook Park, Dong-Min Kim, and Kye-Heon Oh*


The aim of this work was to examine the characteristics and functional activities of wine yeast Pichia species. Initially, two yeasts were isolated from crushed grapes. Using 16S rRNA sequencing, two strains could be assigned to Pichia manshurica and Pichia terricola. Physiological and biochemical characteristics of strain MH-2 was examined. During the incubation period, several physiological activities (e.g., angiotensin-converting enzyme inhibition, tyrosinase inhibitory activity, SOD-like activity, DPPH inhibition) of two strains were evaluated and compared. Based on the results, two wine yeast, P. manshurica and P. terricola might be included as probiotics, presenting the potential to be used in the functional foods for human as well as animal health.

E020

Karyopherins Involved in the Nuclear Actin Transport

Immanuel Dhanasingh and Sung Haeng Lee*

Department of Cellular and Molecular Medicine, Chosun University School of Medicine

Although compartmentalization of a eukaryotic cell into the nucleus and cytoplasm has benefits, it further necessitates transport between the two compartments. Active transport between these two compartments requires metabolic energy and transporter proteins. Nuclear transporter receptors belong to the karyopherin super family and aid in the movement of cargo into (importins) or out of the nucleus (exportins) or are bidirectional. Presence of actin in nucleus and its nuclear trafficking remains intriguing due to its lack of nuclear import or export signals. Despite the fact that importin-9 and exportin-6 have been linked to nuclear import and export of actin respectively, the mechanistic details about actin-karyopherin interaction are not known yet. We compared the structural details of already available karyopherins and provide the initial structural prediction for exportin-6. The WH2 domain from diverse actin binding proteins display a similar architecture of N-terminal α helix followed by the LKKT(V) motif. A similar LKPS motif was identified near helix 14A of exportin-6, which might be the binding site for actin. Based on these predictions, we have postulated a mechanism of actin-karyopherin interaction.


52 | www.msk.or.kr

E021

Crystal Structure of Glycogen Branching Enzyme from Pyrococcus horikoshii

Soo Hui Na and Nam Chul Ha*

Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Seoul National University

Glycogen branching enzyme (GBE) is a branching enzyme that catalyzes the formation of α1,6-branching points during glycogenesis by cleaving α1,4 bond and making a new α1,6 bond. Here, we determine the crystal structure of GBE from the hyperthermophilic archaea, Pyrococcus horikoshii, and PhGBE belongs to glycoside hydrolase 57 (GH57). The crystal of GBE belongs to P21 space group with unit cell dimensions of a = 121.517 Å, b = 43.021 Å and c = 122.682 Å. The final structure of GBE was refined at a resolution of 2.0 Å with and R factor of 18.7% and an R free of 24.7%. The enzyme has a central (β/α)7 fold catalytic domain and an α-helix-rich C-terminal domain, which participates in the formation of the active-site cleft. We found vanillyl acetone as a activator candidate for this enzyme in Natural Compound Library.

E022

Crystal Structure of the Regulatory Domain of AphB, a Virulence Gene Activator from Vibrio vulnificus

Nohra Park, Saemee Song, Inseong Jo, Sang Ho Choi*, and Nam-Chul Ha*

Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University

Many pathogenic bacteria increases expression of virulence factors by recognizing the host environments.Transcriptional activator AphB has been known as a thiol-based switches to sense the anoxic host environment and turn on the transcription of virulence genes in V. vulnificus and Vibrio cholerae. Crystal structures of the full-length AphB from V. cholerae was determined, reporting the tetrameric assembly of AphB whose protomer adopts the typical structures of the LysR-type family proteins consisting of the DNA binding domain and the regulatory domain (RD). The conserved cysteine residue in RD has implicated that it senses the oxygen very sensitively and plays a key role in keeping the structure of AphB at the inactive state. In this study, we determine the crystal structure of AphB RD at 1.9 Å resolution under aerobic conditions. The structure reveals that the conserved cysteine residue is buried in the interior region and does not undergo any oxidation or modification under the aerobic condition. Ensuing biochemical studies also shows that the cysteine residue barely react with the environmental stresses such as hydrogen peroxide, nitric oxide, and alkyl hydrogenperoxide. Our findings suggests that the cysteine residue might not be important in sensing the environmental changes during the pathogenesis. Further studies are required to elucidate the molecular mechanism.

E023

Bacillus licheniformis Contains Two More PerR-like Proteins in Addition to PerR, Fur and Zur Orthologues

Yoon Mo Yang, Jung Hoon Kim, Su Hyun Ryu, Yeh Eun Lee, and Jin Won Lee*

Department of Life Science and Research Institute for Natural Sciences, Hanyang University

The ferric uptake regulator (Fur) family proteins include sensors of Fe (Fur), Zn (Zur), and peroxide (PerR). Among Fur family proteins, Fur and Zur are ubiquitous in most prokaryotic organisms, whereas PerR exists mainly in Gram positive bacteria as a functional homologue of OxyR. Gram positive bacteria such as Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus encode three Fur family proteins: Fur, Zur and PerR. In this study, we identified five Fur family proteins from B. licheniformis: two novel PerR-like proteins (BL00690 and BL00950) in addition to Fur (BL05249), Zur (BL03703), and PerR (BL00075) homologues. Our data indicate that all of the five B. licheniformis Fur homologues contain a structural Zn2+ site composed of four cysteine residues like many other Fur family proteins. Furthermore, we provide evidence that the PerR-like proteins (BL00690 and BL00950) as well as PerRBL (BL00075), but not FurBL (BL05249) and ZurBL (BL03703), can sense H2O2 by histidine oxidation with different sensitivity. We also show that PerR2 (BL00690) has a PerR-like repressor activity for PerR-regulated genes in vivo. Taken together, our results suggest that B. licheniformis contains three PerR subfamily proteins which can sense H2O2 by histidine oxidation not by cysteine oxidation, in addition to Fur and Zur.[This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) [NRF-2011-0017199].]

E024

Effect of Exogenous Glutamine on Salmonella Replication under Nitrosative Stress Conditions

Yoon Mee Park and Iel Soo Bang*

Department of Microbiology and Immunology, Chosun University School of Dentistry

Nitric oxide (NO) damages various bacterial macromolecules, resulting in abnormal metabolism and subsequent growth arrest. But, the NO targets and strategies for avoiding nitrosative stress in bacteria are still not largely understood In previous studies, we have shown that NO can cause auxotrophy for amino acids in hmp mutant S. Typhimurium that can hardly metabolize NO entering into bacteria. This study presents that exogenous supplementation with glutamine (Gln) or glutamate (Glu) can fully restore the replication of hmp mutant S. Typhimurium in NO-producing cultures. Because reactions for the biosynthesis of Gln and Glu are interrelated, we analyzed the effect of supplementation with Gln or Glu on the growth of hmp mutants further lacking genes for the biosynthesis of Gln and/or Glu, and found that, in the presence of NO, Salmonella required Gln for their growth. Only L-form of Gln could restore bacterial growth, suggesting that this growth recovery may have less relation with bacterial cell wall biosynthesis. The Gln supplementation did not affect the NO consumption rate of wild type and hmp mutant S. Typhimurium, indicating that the Gln-promoted NO resistance is independent of NO metabolism itself. These results suggest that cytotoxic NO may impair reactions for maintaining levels of Gln in S. Typhimurium, resulting in auxotrophy for Gln, but which can be overcome by bacterial taking up Gln from surroundings. [Supported by NRF grant (2008-0062283)]

Poster

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E025

Crystal Structure of Bacterial 1-Cys Peroxiredoxin from Vibrio vulnificus and Its Structural and Functional Implications to Scavenging ROS and Nitric Oxide

Jinsook Ahn1, Kyung Ku Jang1, Inseong Jo1, Jin-Wook Yoo2, Sang Ho Choi1, and Nam-Chul Ha1*1Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, Research Institute for Agricultural and Life Sciences, Seoul National University, 2Department of Manufacturing Pharmacy, Pusan National University

Peroxiredoxins are the ubiquitous cysteine-based peroxidase enzymes. Recently, a new type Peroxiredoxin, called VvPrx3, was identified in a pathogenic bacterium Vibrio vulnificus as an essential gene for survival in macrophage. It belongs to 1-Cys peroxiredoxin family that contains only one catalytic cysteine residue (Cys48). Interestingly, VvPrx3 was induced in response to nitric oxide (NO) treatment, different from other types of Prx in V. vulnificus. Here, we determine the crystal structures of VvPrx3 from V. vulnificus both in the reduced and the oxidized forms at high resolutions. The crystal structure in the reduced form showed a typical dimeric interface (A-type contact), and the structure at the oxidized state revealed a novel oligomeric interface, called C-type contact, between the typical dimer. The C-type contact of VvPrx3 is mediated by a disulfide bond between the catalytic cysteine residues and induced by treatment of peroxides. Ensuing studies showed that the disulfide bond of VvPrx3 is also made by the NO treatment, and VvPrx3 is important in survival of the bacteria in the presence of NO. Taken together, we propose a novel function of 1-Cys peroxiredoxins in direct scavenging of NO stress via a novel type of oligomerization, and our findings would help understand diverse functions of peroxiredoxins during pathogenic process at the molecular level. [Supported by grants from SHC and NCH]

E026

The HPr-pyruvate Kinase Complex Protects Vibrio vulnificus Cells against H2O2 Stress Generated by Fungal Neighbors in the Presence of Glucose

Hey-Min Kim1, Young-Ha Park1, Chang-Kyu Yoon1, and Yeong-Jae Seok1,2*1Department of Biological Sciences and Institute of Microbiology, 2Department of Biophysics and Chemical Biology, Seoul National University

We recently reported that dephospho-HPr increases the cellular level of pyruvate by interacting with pyruvate kinase A (PykA) in Vibrio vulnificus, but not in Escherichia coli. Here, we show that this interaction confers cell survival under H2O2 stress by stimulating PykA-mediated pyruvate production in the presence of glucose. A pykA deletion mutant was more susceptible to H2O2 than wild-type V. vulnificus despite no significant difference in the expression level of catalase, and there was a more remarkable difference of H2O2 sensitivity between wild-type and pykA mutant strains in the presence of glucose than galactose. Because some fungi are known to produce hydrogen peroxide through the reaction catalyzed by glucose oxidase, we tested whether fungi isolated from the natural habitat of V. vulnificus exert an inhibitory effect on V. vulnificus survival. While Aspergillus fumigatus did not produce H2O2 and thus its culture filtrate did not kill V. vulnificus cells regardless of the sugar source, A. welwitschiae produced H2O2 and showed bactericidal activity only in the presence of glucose. The pykA mutant showed more severe growth retardation than wild-type V. vulnificus in the culture filtrate of glucose-grown A. welwitschiae and this sensitivity was rescued by the addition of pyruvate. These data suggest that V. vulnificus resists the killing activity of its fungal neighbors by increasing pyruvate production in the presence of glucose. [Supported by NRF]


54 | www.msk.or.kr

F001

Complete Genome Analysis of Vibrio parahaemolyticus FORC_023, Isolated from Storage Water for Raw Fish

Han Young Chung, Byungho Lee, Eun Jung Na, and Sang Ho Choi*

Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Seoul National University

Vibrio parahaemolyticus is a Gram-negative bacterium and consumption of seafood contaminated with this bacterium can cause gastroenteritis. V. parahaemolyticus FORC_023 strain was isolated from the storage water for raw fish, and its whole genome was sequenced. The genome FORC_023 is composed of two circular chromosomes without plasmid. Chromosomes consist of 4,227 predicted open reading frames (ORFs), 131 tRNA genes, and 37 rRNA genes. Among various V. parahaemolyticus, FORC_023 shows the highest average nucleotide identity (ANI) value with UCM-V493 which is one of the most virulent strains, indicating that FORC_023 is virulent as well. Furthermore, comparative genome analysis of the two strains revealed that FORC_023 carries an additional genomic region encoding strong virulence factors such as repeats-in-toxin (RTX) and type II secretion, which could lead to host cell death. In addition, in vitro cytotoxicity testing suggests that FORC_023 exhibits a higher level of cytotoxicity toward the INT-407 human epithelial cells as determined by lactose dehydrogenase (LDH) release. All these results suggest that the FORC_023 isolated from storage water for raw fish is one of the most virulent strains of V. parahaemolyticus, providing new insights into the necessity for rapid detection as well as epidemiological investigation to prevent food-borne outbreaks by V. parahaemolyticus. [This research was supported by a grant (14162MFDS172) from Ministry of Food and Drug Safety in 2015.]

F002

Complete Genome Sequence of Antibiotic and Anticancer Agent Violacein Producing Massilia sp. Strain NR 4-1

Nu Ri Myeong, Hoon Je Seong, Hye-Jin Kim, and Woo Jun Sul*


Massilia sp. NR 4-1 was a violacein producing strain newly isolated from topsoil under nutmeg tree, Torreya nucifera in Korean national monument Bijarim Forest, Jeju. Massilia sp. NR 4-1 was identified as producing the violet pigments (violacein and deoxyviolacein). Violacein is a novel class of drug exhibiting anticancer and antibiotic activities originated from L-tryptophan. Here, we present the complete genome of Massilia sp. strain NR 4-1 of 6,361,416 bp and total 5,285 coding sequences (CDSs) including a complete violacein biosynthesis pathway, vioABCDE. Presence of five genes involved in violacein production pathway was identified. The genome sequence of Massilia sp NR 4-1 will provide stable and efficient biotechnological applications of violacein production.

F003

Metabolic Role of MADS-box Transcription Factor Mbx2 in Schizosaccharomyces pombe

Youngdae Seo and Jung-Hye Roe*


In eukaryotes, glucose is mainly assimilated through the respiration pathway in mitochondria for maximum energy yield in the presence of oxygen. However, yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe undergo aerobic fermentation in which glucose is predominantly fermented to ethanol even in the presence of oxygen. In this study, we examined the role of a putative transcription factor Mbx2 with MADS-box in energy metabolism. Growth of mbx2Δ in minimal media was monitored by spectrophotometer. The doubling time and the final OD of mbx2Δ increased about 30% and decreased about 30%, respectively, compared with the wild type. The entry time point of stationary growth phase was similar between the wild type and the mutant. The amount of glucose in cell cultured media, however, was not depleted at the same time. Whereas the wild type consumed almost all the glucose by the onset of stationary phase, glucose in the mutant culture media was depleted far after entering the stationary phase. The glucose uptake level per cell, however, showed little difference between the wild type and the mutant. Measurement of the respiration rates throughout growth phases revealed that the oxygen consumption rate was reduced by about 30% in the mutant, compare with the wild type. The mechanism behind this metabolic function is being investigated.

F004

Role of Rsv1 in Responding to Glucose-starvation in Schizosaccharomyces pombe

Eun-Jung Kim and Jung-Hye Roe*


In natural environment cells are faced with various conditions that cause metabolic stresses. Metabolic stresses are caused by diverse nutrients depletion, and among the nutrients glucose is critical as an energy and carbon source. Cells can adjust their physiological states upon glucose shortage in transcriptional and post-translational levels. In the fission yeast Schizosaccharomyces pombe, Rsv1 protein with C2H2-type zinc finger domain acts as a transcription factor to maintain cellular physiological function in glucose-starved condition. Rsv1 protein is expressed under low glucose condition, but not under glucose-depleted (no glucose) condition. When rsv1 gene-deleted mutant cells are transferred to low glucose media, cells show lower CFU (colony forming units) than wild-type cells on solid media. Moreover, Δrsv1 mutants lose their resistance to other stresses like oxidative and ethanol stresses. We isolated putative interacting partners of Rsv1 protein through immunoprecipitation-based LC-MS/MS experiment. Candidates include some protein kinases, ATPases, snRNP complex subunits, RNA-binding proteins, etc. Through PTM (post translational modification) analysis, we found some phosphorylated or ubiquitinated amino acids in Rsv1 protein. We predict some responsible kinases and reveal possible signal transduction pathways to activating Rsv1 function.

Poster

www.msk.or.kr | 55

F005

A Genome-wide Transcriptomic Analysis of Radiation-responsive Genes in the Radiation-resistant Fungus, C. neoformans

Kwang-Woo Jung1, Min-Kyu Kim1, Dongho Kim1, Sangyong Lim1, and Yong-Sun Bahn2*1Research Division for Biotechnology, Korea Atomic Energy Research Institute2Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University

The basidiomycetous fungus Cryptococcus has been known as radiation resistant fungi and is found in highly radioactive environments such as the damaged nuclear reactor at Chernobyl. Although Cryptococcus exhibits greater resistant for gamma radiation than model yeast Saccharomyces cerevisiae, the resistant mechanism of gamma radiation remains elusive. To elucidate a unique regulatory system for radiation-resistance in C. neoformans, we performed genome-wide comparative analysis through DNA microarray analysis using C. neoformans WT strain (serotype A, H99 strain) responding gamma radiation. Based on the transcriptome analysis, genes involved in DNA damage repair systems (RAD51, RDH54, and RAD54) were significantly increased in response to gamma radiation. Actually, rad54Δ and rdh54Δ mutants exhibited sensitivity against both gamma radiation and DNA damage inducers. Furthermore, genes regarding to molecular chaperone and ubiquitination systems were strongly induced. In contrast, expression levels of genes related to protein synthesis, fatty acids/sterols synthesis, and other cellular molecules. Especially, ergosterol homeostasis is required for gamma radiation resistance. Furthermore, radiation-induced genes such as RIG4, RIG5, and RIG6 in C. neoformans play critical roles in gamma radiation resistance. Taken together, the transcriptome analysis contributes to understanding unique molecular mechanism of radiation-resistant fungus C. neoformans.

F006

Crystal Structure of Thermoplasma acidophilum XerA Recombinase Shows Large C-shape Clamp Conformation and cis-cleavage Mode for Nucleophilic Tyrosine

Chang Hwa Jo1, Junsoo Kim1, Ah-reum Han1, Sam Yong Park2, Kwang Yeon Hwang1, and Ki Hyun Nam3*1Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University, 2Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Japan, 3Pohang Accelerator Laboratory, Pohang University of Science and Technology

The Xer recombinase, a site-specific tyrosine recombinase, plays an important role in a variety of biological processes, including excisional recombination of plasmid DNA, and resolution of catenated DNA circles and host chromosome DNA. Xer monomers bind to two double-stranded DNA dif sites and mediate breaking, exchange, and rejoining of four DNA phosphodiester bonds. Here we report the 2.5 Å crystal structure of XerA from the archaean Thermoplasma acidophilum. Crystallographic data reveal a uniquely open conformational state, resulting in a C-shaped clamp with an angle of ~48° and a distance of 57 Å between the core-binding and the catalytic domains. The catalytic nucleophile, Tyr264, is positioned in cis-cleavage mode by XerA’s C-term tail that interacts with the CAT domain of a neighboring monomer without DNA substrate. Structural comparisons of tyrosine recombinases elucidate the dynamics of Xer recombinase.[This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2014R1A1A2059440 and 2013R1A1A2008404)]

F007

Oxidative Stress Response of Deinococcus geothermalis via a Cystine Importer

Minwook Kim and Sung-Jae Lee*

Department of Biology, Kyung Hee University

The responses to oxidative stress induced by H2O2 of the wild-type and a dgeo_1985 gene-disrupted mutant strain of Deinococcus geothermalis were phenotypically different. At a certain concentration of H2O2, the ∆dgeo_1985R strain was more resistant to oxidative stress than was the wild-type strain, as determined using bacterial growth and viability assays. The wild-type and mutant strains expressed equal levels of superoxide dismutase and catalase under H2O2-induced stress. Although the expression levels of the general DNA-damage response-related genes recA, pprA, ddrA, and ddrB were up-regulated by more five-fold in the wild-type strain relative to those in the ∆dgeo_1985R mutant strain, the mutant strain had a higher survival rate than that of the wild-type when various types of stress were induced. A cystine-transporter gene (dgeo_1986) was highly expressed in the ∆dgeo_1985R mutant strain, at a level that was greater than 150-fold that of the wild-type strain, leading to the conclusion that this cystine transporter might be involved in the defensive response to H2O2 stress. In this study, the cystine transporter was identified and characterized through membrane protein-expression analysis, a cystine-binding assay, and assays of the intracellular H2O2, cysteine, and thiol levels. Therefore, this cystine-uptake system functions as a primitive defense system that non-enzymatically scavenges oxidative stress agents in D. geothermalis.

F008

Complete Genome Sequence of Vibrio parahaemolyticus FORC_022, a Food-borne Pathogen from Soy Sauce Marinated Crab in South Korea

Byungho Lee, Han Young Chung, Suyeon Kim, Eun Jung Na, and Sang Ho Choi*

Foodborne-pathogens Omics Research Center, Department of Food Science and Biotechnology, Seoul National University

Vibrio parahaemolyticus is a curved rod-shaped bacterium that causes gastrointestinal illness associated with the consumption of seafood. To characterize V. parahaemolyticus that have caused outbreaks in South Korea, ten strains of V. parahaemolyticus were obtained from Korean Ministry of Food and Drug Safety. Virulence gene-specific PCR screening and lactate dehydrogenase (LDH) release assay were employed to evaluate the virulence of these strains. Among strains, the H8 strain, which was isolated from a crab marinated with soy sauce, had the many virulence genes and the strain showed a high level of cytotoxicity in the LDH release assay. Therefore, the H8 strain was selected for genome sequencing and was designated as FORC_022. The FORC_022 genome consists of two chromosomes and a plasmid, which have the GC content of 45.2%, 45.37%, and 44.5%, respectively. The genome was predicted to have 4,858 open reading frames, 133 tRNA genes, and 37 rRNA genes. Average nucleotide identity analysis using nine complete V. parahaemolyticus genome sequences revealed that FORC_022 was closely related to clinical strains. Furthermore, virulence factors of FORC_022 were identified using BLAST against Virulence Factor Database. This study will deepen our understanding of the pathogenesis of V. parahaemolyticus and help improve the methods in detection of the bacterium in foodborne outbreaks. [This research was supported by a grant (14162MFDS972) from Ministry of Food and Drug Safety in 2016.]


56 | www.msk.or.kr

F009

Transcriptome Analyses of a Novel Transcriptional Regulator HpxA Essential for Hypoxic Responses in Aspergillus nidulans Using RNA-Seq

Sun-Ki Koh, Dawoon Chung, Jun-Yong Kwak, Mee-Hyang Jeon, and Suhn-Kee Chae*

Department of Biochemistry, Paichai University

Hypoxia-sensitive hpxA mutant alleles and ΔhpxA in Aspergillus nidulans showed completely blocked growth in hypoxia (1% O2) and significantly increased susceptibility to an antifungal drug itraconazole compared to wild type (WT). These phenotypes were consistent with the null-mutant phenotypes of srbA encoding a sterol regulatory element binding protein (SREBP), a transcriptional regulator critical for hypoxia adaptation in fungi. To investigate genes whose transcriptions are regulated by HpxA, we performed RNA sequencing analyses with ΔhpxA and WT strains under hypoxic conditions. A total of 183 genes were down-regulated in ΔhpxA by more than 4-fold relative to WT, while 300 genes were up-regulated in ΔhpxA. Interestingly, srbA transcription levels were decreased in ΔhpxA compared to WT, and several SrbA regulons involved in oxygen-consuming processes including syntheses of ergosterol, heme, and coenzyme Q10 were also identified as HpxA target genes. To elucidate a link between HpxA and SrbA, we conducted quantitative real-time PCR with ΔhpxA and ΔsrbA in hypoxia. Compared to WT, expression of hpxA was increased in ΔsrbA by about 3-fold, while expression of srbA was reduced in ΔhpxA by about 0.6-fold. Our study suggests the presence of a putative linear genetic relationship between two transcriptional regulators for hypoxia adaptation in A. nidulans. [Supported by the NRF (No. 2015R1D1A1A01057677)]

F010

Yap1 and Skn7 are Involved in DNA Double-strand Break Repair by Homologous Recombination in Saccharomyces cerevisiae

Myung Ju Kim1,2, Dae Gwan Yi3, Jihyun Lee1,2, Ji Eun Choi1,2, Bohyun Park1, Sujin In1, and Woo-Hyun Chung1,2*1College of Pharmacy, 2Innovative Drug Center, Duksung Women's Universiity3Department of Biological Sciences, Seoul National University

Cells are continually exposed to numerous exogenous and endogenous agents that cause DNA damage. Although substantial proportion of lethal DNA damage is attributed by direct DNA strand breaks, oxidative DNA adducts induced by reactive oxygen species (ROS) are also significant sources of mutagenesis and genomic instability that might eventually lead to tumorigenesis as well as aging. Pathways of DNA damage repair and oxidative stress responsive signaling have been proposed to be highly associated in the cell, but the underlying molecular mechanism remains unknown. We employed mutant strains lacking Rad51, the homolog of E. coli RecA recombinase, and Yap1 or Skn7, two major transcription factors responsive to ROS, to examine genetic interactions between double-strand break (DSB) repair proteins and cellular redox regulators in budding yeast Saccharomyces cerevisiae. Abnormal expression of YAP1 or SKN7 aggravated spontaneous mutation rate and sensitivity of rad51 mutant to DSB- and ROS-generating reagents. Rad51 deficiency contributed to genome instability more in response to the increased ROS, and the accumulation of DSB lesions raised the intracellular ROS level. Our findings suggest that there is a significant crosstalk between DSB repair pathways and ROS signaling proteins for cell survival and maintenance of genome stability in response to genotoxic stresses.[Supported by grant from KRF]

F011

Role of Conserved Residues within the Wedge Domain of Deinococcus radiodurans RecG

Sun Wook Jeong, Jing Zhang, Lei Zhao, Min Kyu Kim, and Sangyong Lim*

Research Division Biotechnology, Korea Atomic Energy Research Institute

Deinococcus radiodurans has exceptional ability to repair DNA damage caused by various DNA-damaging agents including ionizing radiation (IR). Some deinococcal Rec proteins are known to be different from counterparts of the IR-sensitive bacterium Escherichia coli in structure and function. RecG helicase is highly evolutionarily conserved in bacteria and required for efficient DNA repair. The comparison of amino acid sequence revealed that three amino acids ‘201QPW203’ located in the wedge domain responsible for DNA binding are well conserved in Deinococcus RecG. We constructed the DrRecG derivative DrRecGFSA, in which ‘QPW’ was substituted by the equivalent of ‘99FSA101’ in EcRecG, and the EcRecG derivative EcRecGQPW, in which ‘FSA’ was substituted by ‘QPW’. EcRecGQPW partially complemented ΔrecG in trans under MMC-stress conditions, whereas DrRecGFSA did not complement ΔrecGDR at all like EcRecG. DNA binding activities of the purified recombinant wedge domains of DrRecG and EcRecGQPW were higher than that of EcRecG. We further examined DNA repair kinetics in ∆recGDR containing RecG derivatives and found that the genome shattered by IR was readily reassembled in ∆recGDR containing DrRecG and EcRecGQPW. These results suggest that the QPW residues facilitate strong binding of DrRecG to DNA junctions, thereby enhancing the efficiency of DrRecG in DNA repair process.

F012

Complete Genome Sequencing of Clinical Isolated Salmonella enterica FORC_020 and Comparative Genome Analysis with S. enterica Typhimurium LT2 and S. enterica Newport USDA-ARS-USMARC-1972

You-Tae Kim and Ju-Hoon Lee*

Department of Food Science and Biotechnology, Institute of Life Sciences and Resources, Kyung Hee University

Salmonella enterica is a well-known food-borne pathogen causing salmonellosis according to what serotype could be harmful to human. S. enterica FORC_020 (NCCP P3567) is a clinical isolate from a blood sample of a food-poisoned patient. To determine the serotype and understand its pathogenicity and virulence mechanism for food poisoning in genomic level, its genome was completely sequenced using hybrid sequencing and analyzed using various bioinformatic programs. The complete genome size of strain FORC_020 was 4,799,793 bp with GC content of 52.26%. It contains 83 tRNA genes, 7 rRNA operons, and a single additional 5S rRNA gene. The genome was annotated to 4,671 open reading frames (ORFs) consisting of 3,957 ORFs predicted to encode functional proteins. Phylogenetical location of strain FORC_020 is closest to S. enterica Typhimurium LT2 in 16S rRNA sequence analysis with >99% DNA sequence identity by BLASN program. In Average Nucleotide Identity (ANI) analysis and Multilocus Sequence Typing (MLST), meanwhile, strain FORC_020 was located close to Newport Serotype and belonged to Newport of MLST database. Comparative genomics was performed using ACT and MAUVE and two large different regions were detected. One is for inner membrane protein clusters and the other is for proteins related to DNA sulfur modification. This region is unique in Salmonella as well as other bacteria. [Supported by grants from MFDS]

Poster

www.msk.or.kr | 57

F013

ABC Transporter Atm1 Plays Roles in Mitochondrial Functions and Iron Homeostasis in Human Fungal Pathogen Cryptococcus neoformans

Eunsoo Do, Se-Ho Park, and Won Hee Jung*


Iron sulfur cluster (ISC) is an indispensable cofactor for various enzymes, including mitochondrial respiratory chains, DNA repair, transcription and many catalytic enzymes. In addition, in the model yeast Saccharomyces cerevisiae, the mitochondrial ABC transporter, Atm1, has an important role in exporting the unknown substrate for cytosolic ISC assembly machinery for the cytosol and nucleus ISC containing proteins. In this study, we identified an ortholog of S. cerevisiae Atm1 in the human fungal pathogen Cryptococcus neoformans. To characterize the functions of Atm1 in C. neoformans, the strain lacking ATM1 gene was constructed, and its phenotypes related to mitochondrial functions and iron homeostasis were investigated. Our results showed that activity of mitochondrial superoxide dismutase Sod2 was diminished and that cellular ROS was highly increased in the atm1 mutant, which may be caused by distorted mitochondrial ISC homeostasis in the mutant cells. Furthermore, we found that the atm1 mutant displayed increased intracellular iron level compared with wild-type. Overall, our results suggested that Atm1 influences mitochondrial functions and plays a role in iron homeostasis in C. neoformans. [This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning NRF-2013R1A1A1A05007037]

F014

Distinct Survival Strategies of Pseudomonas aeruginosa by Dual Promoters of the Major Catalase (KatA)

In-Young Chung, Bi-o Kim, Hye-Jung Jang, and You-Hee Cho*

Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Sciences, CHA University

KatA is the major catalase required for hydrogen peroxide resistance and acute virulence in Pseudomonas aeruginosa PA14, whose transcription is driven from the promoter (katAp1) located at 155 nucleotide (nt) upstream of the start codon. Here, we identified another promoter (katAp2), the +1 of which was mapped at the 51 nt upstream of the start codon, which was responsible for the basal transcription during the planktonic culture and down-regulated upon H2O2 treatment under the control by the master regulator of anaerobiosis, Anr. To dissect the roles of the dual promoters in conditions involving KatA, we created the promoter mutants for each -10 box (p1m, p2m, and p1p2m) and found the separate roles of the dual promoters to cope with reactive oxygen and nitrogen species (ROS and RNS), which is dependent on the growth states. The katAp1 is required for the function of KatA in the logarithmic growth phase as well as in acute virulence, whereas katAp2 is required for the function of KatA in the stationary phase during the planktonic culture as well as in the prolonged biofilm culture. This dismantling of dual promoter regulation of katA sheds light on the roles of KatA in response to both proliferating and growth-restricting conditions and thus provides an insight into their metabolic impacts on the survival strategies of P. aeruginosa.

F015

Functional Analysis of RraAS1 Interacting with the Catalytic Domain of RNase ES

Daeyoung Kim, Sojin Seo, Boeun Lee, Minju Joo, Ji-Hyun Yeom, and Kangseok Lee*


RNase E is an essential enzyme that participates in processing and degradation of RNAs in Escherichia coli. Enzymatic activity of RNase E is controlled by a regulator of ribonuclease activity, RraA and RraB. The gram positive bacterium Streptomyces coelicolor also contains homologs of RNase E and RraA, designated as RNase ES, RraAS1 and RraAS2. In this study, we investigated the inhibitory function of RraAS1 on ribonucleolytic activity of RNase ES. Unlike other RraA homologs, which normally require both of the catalytic and scaffold domains, RraAS1 inhibited the enzymatic activity of RNase ES in vivo and in vitro by interacting with the catalytic domain of RNase ES, Coexpression of RraAS1 reduced the ribonucleolytic activity in cells overproducing RNase ES and consequently rescued these cells from growth arrest. Analyses of steady-state level of several RNase E substrates indicated that the coexpression of RraAS1 efficiently inhibited RNase ES action and resulted in the increased abundance of each RNA species. Intriguingly, deletion of rraAS1 in Streptomyces coelicolor resulted in early sporulation and hyper-production of actinorhodin antibiotic compared with wild type strain. These results suggest that RraAS1 affects the global RNA stability by regulating enzymatic activity of RNase ES in Streptomyces coelicolor.

F016

Functional Analysis of RraAS2, a Streptomyces coelicolor Homolog of RraA

Jihune Heo, Sojin Seo, Boeun Lee, Daeyoung Kim, Minju Joo, Ji-Hyun Yeom, and Kangseok Lee*


RraA is a protein inhibitor of RNase E, which catalyzes the endoribonucleolytic cleavage of a large proportion of RNAs in Escherichia coli. RraA has also been shown to regulate the activity of RNase ES, a functional Streptomyces coelicolor ortholog of RNase E, which has 58.0% identity to the amino-terminal catalytic region of RNase E. The antibiotic-producing bacterium Streptomyces coelicolor also contains homologs of RraA, designated as RraAS1 and RraAS2. Here, we report that RraAS2 inhibits the endoribonucleolytic activity of RNase ES in vivo as well as in vitro; however, unlike other RraA homologs that require the C-terminal scaffold domain to exert their inhibitory effect, RraAS2 required both of two concurrent domains of the scaffold region and the catalytic domain of RNase ES to do so. We further show that deletion of the RraAS2 gene in S. coelicolor results in hyper-production of actinorhodin antibiotic and a higher growth rate compared with wild type strain. These findings suggest that RraAS2-mediated regulation of RNase ES activity contributes to modulating the cellular physiology of S. coelicolor.


58 | www.msk.or.kr

F017

RNase G Modulates Pathogenicity of Salmonella Typhimurium through the Control of hns mRNA Abundance

Hong-Man Kim1, Daeyoung Kim1, Minho Lee1, Ji-Hyun Yeom1, Boeun Lee1, Wooseok Song2, and Kangseok Lee1*1Department of Life Science, Chung-Ang University2Department of Microbiology, Catholic University of Daegu, School of Medicine

Bacterial ribonucleases regulate gene expression through RNA processing and decay. Among them, RNase G is an endoribonucleases, which is involved in rRNA processing and degradation of a few mRNAs in Escherichia coli. However its physiological role remains largely uncharacterized. Here, we report that RNase G (Rng) controls expression levels of histone-like nucleoid structuring protein (H-NS) encoded by hns, which were strongly associated with the pathogenicity of S. Typhimurium cells in both epithelial cells and mice. We observed that RNase G expression in S. Typhimurium cells was induced when they were exposed to high-salt condition and infected into epithelial cells, and coincidentally expression levels of hns was decreased. We further demonstrated that expression levels of Salmonella virulence regulators, hilA, sipA, and hns, were tightly regulated by RNase G in anoxic high-salt and infection conditions, which has a correlation with S. Typhimurium pathogenicity. In addition, we validated that hns mRNA abundance is mediated by RNase G, where 5’UTR of hns mRNA was directly cleaved by RNase G in vivo and in vitro. In conclusion, we suggest that RNase G-mediated modulation of Salmonella pathogenicity island 1 type III secretion system involves H-NS as a key factor for the survival and virulence of S. Typhimurium in host cell.

F018

Change of Biochemical Characteristics in aroA ompA Deletion in Salmonella enterica serovar Enteritidis

Kiju Kim and Tae-Wook Hahn*

College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University

Salmonella enterica serovars Enteritidis (SE) is one of the well-known foodborne pathogen causing salmonellosis in the world. Many attenuated Salmonella mutants were developed for using vaccine strain against salmonellosis and live attenuated Salmonella strains can be powerful vaccine candidates for expression of heterologous antigen to the mammalian immune system. In particular, antigen delivery using a Salmonella strains can enhance mucosal immunity as well as systemic immune responses. We constructed an aroA ompA deletion SE mutant using the phage λ-red recombinase system. The biochemical characteristics of SE mutant were identified by the Vitek II system (bioMérieux, France) based on Gram-negative card according to manufacturer's instructions. The SE aroA ompA mutant resulted in an inability to produce H2S production (H2S), lipase (LIP) and alpha-galactosidase (AGAL) compared with SE wild type. Interestingly, we found the expression of another enzymes such as gamma-glutamyl- transferase (GGT), L-lactate alkalinisation (ILATk) and phosphatase (PHOS) in SE aroA ompA mutant. These findings indicate that SE aroA ompA mutant may regulate to express different enzymes by unknown biochemical mechanism.

F019

Molecular Mechanism of Anti-repressor by Ler to LEE5 Promoter in Enteropathogenic Escherichia coli (EPEC)

Minsang Shin1 and Hyon E. Choy2*1Department of Microbiology, Kyungpook National University School of Medicine2Department of Microbiology, Chonnam National University Medical School

Secretion of effector proteins in Enteropathogenic Escherichia coli (EPEC) and Enterohemorrhagic Escherichia coli (EHEC) is mediated by a specialized type III secretion system which components are encoded in the LEE1-5 operons. H-NS, a global repressor in E. coli, silences the expression of LEE operons. Ler, a master regulator in LEE operons, shares 24% amino acid identity and 44% amino acid similarity to H-NS. Interestingly, rather than a gene silencer, its main role has been characterized as an antagonizing protein that relieves H-NS-mediated transcriptional silencing. In the previous study we reported molecular mechanism for the repression of LEE5p in EPEC and EHEC by H-NS as a protein–protein interaction between upstream DNA-bound H-NS and the αCTD of promoter-bound RNAP. The mechanism of Ler in alleviating genes repressed by H-NS is largely unknown. We examined binding affinity by both regulator at LEE5 promoter using a various in vitro work. Our result show that binding affinity of Ler to the LEE5p DNA is about 40 folds greater than that of H-NS using surface plasmon resonance technology. Ler binding should remove H-NS binding to the same stretch of DNA.[Supported by grants from NRF-2014R1A2A1A10051664]

F020

Cloning and Expression of 5-aminolevulinic Acid Synthase Gene Involved in Secondary Metabolism of Kitasatospora cheerisanensis

Hwang Jae Yoon and Doo Hyun Nam*

College of Pharmacy, Yeungnam University

Recently the biosynthetic pathway for the formation of 2-amino-3- hydroxycyclopent-2-enone (C5N) unit and its attachment to core structure of secondary metabolites in actinomycetes has been revealed. It has been revealed that three enzymes, 5-aminolevulinate synthase (ALAS), amino-levulinate-CoA ligase (ALL) and amide synthetase (AMS) are involved in the formation and attachment of C5N unit in secondary metabolite, which appear as a set of encoded genes located within a relevant biosynthetic gene cluster. Among the three genes, the ALAS gene of Kitasatospora cheerisanensis KCTC2395 which is assumed to be responsible for the C5N formation for the biosynthesis of bafilomycin B1 compound was cloned and heterologously expressed in E. coli. The biochemical characterization of recombinant ALAS of K. cheerisanensis was attempted by colorimetric assay of the enzyme activities in this study. The recombinant ALAS protein was confirmed to produce 5-ALA from glycine and succinyl-CoA. The enzyme reaction occurred time-dependently upon the concentration of substrates. The optimal reaction pH was 8.5, and the optimal concentration of pyridoxal-5’-phosphate (PLP) as a cofactor in this reaction system was found to be 0.1 mM.

Keywords: Kitasatospora, 5-aminolevulinate synthase, ALAS, C5N, bafilomycin

Poster

www.msk.or.kr | 59

F021

The Velvet Regulators and Their Targets in Aspergillus nidulans

Hee-Soo Park 1,3*, Pil Jae Maeng2, and Jae-Hyuk Yu3

1School of Food Science and Biotechnology, Kyungpook National University, 2Department of Microbiology and Molecular Biology, Chungnam National University, 3Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA

The NF-kB like fungal transcript factors VosA and VelB are key regulators which control spore viability, maturation and β-glucan synthesis in Aspergillus nidulans. The VosA-VelB hetero-complex directly or indirectly regulates spore-specific genes and development-associated genes. Here, we report various targets of the VosA-VelB hetero-complex. Frist, VosA-VelB controls a β-glucan synthesis in both asexual and sexual spores. Second, VosA-VelB directly regulates trehalose biosynthesis in asexual spores. Third, mRNA accumulation of several developmental genes is controlled by VosA and VelB. Overall, these results support that the VosA-VelB complex is a master transcriptional unit for sporogenesis in Aspergillus nidulans.

F022

Effects of the Number of the Origin of Replication on the Cell Physiology of Escherichia coli

Hee Jin Yang, Yuna Jung, and Jihyun F. Kim*

Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University

Bacteria, having circular genomes in most cases, have a single origin of replication (ori) for each replicon, while archaea and eukaryotes may have multiple origins of replication in each chromosome. Initiation of DNA replication in Escherichia coli is bi-directionally triggered at oriC and arrested at the termination sites (ter). We questioned what if E. coli has multiple origins of replication in its chromosome and how it may affect cell physiology including cell division. We attempted to introduce additional wild-type oriC of E. coli into intergenic regions that should have no direct effect on cell physiology. Consequently, we constructed E. coli derivatives with two or three oriCs which are separated from the original oriC by 1 Mb or 2 Mb. Recombinant E. coli cells exhibited a range of cell-size variations; some appeared indistinguishable from wild type with no morphological abnormalities. Generation time of Recombinant E. coli increased 5 to 10% in a minimal medium with glucose as a sole carbon source, which was not different in several rich media. From the results, some E. coli cells with multiple origins did not cause any defects on their morphology and cell cycle.


60 | www.msk.or.kr

G001

Metabolically Engineered Escherichia coli for Renewable Production of 3-Aminopropionic Acid

Tong Un Chae1, Chan Woo Song1,2, and Sang Yup Lee1,2,3*1Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIS, 2BioProcess Engineering Research Center, KAIST, 3BioInformatics Research Center, KAIST

In this study, a novel metabolic pathway was designed for the production of 3-aminopropionic acid (3-AP) in Escherichia coli. Using a fumaric-acid producing E. coli strain as a host, the C. glutamicum panD gene (encoding L-aspartate-α-decarboxylase) was overexpressed and the native promoter of the aspA gene was replaced with the strong trc promoter, which allowed aspartic acid production through the aspartase (AspA)-catalyzed reaction. Additional overexpression of the aspA and phosphoenolpyruvate carboxylase (ppc) genes, and the supplementation of ammonium sulfate in the medium allowed production of 3.49 g/L 3-AP. This was further increased to 3.94 g/L by optimizing the expression level of PPC, which was achieved by evaluating 12 different combinations of synthetic promoters and RBS sequences. Fed-batch culture of the final strain yielded 17.9 g/L 3-AP in 89 h, with an overall yield and productivity of 0.186 g 3-AP/g glucose and 0.200 g/L/h, respectively. [Development of systems metabolic engineering platform technologies for biorefineries(NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) funded by the Ministry of Education, Science and Technology]

G002

Engineering TCA Cycle for Renewable Production of Fumaric Acid by Escherichia coli

Tong Un Chae1, Chan Woo Song1,2, Dong In Kim1,3, Sol Choi1,2, Jae Won Jang1,2, and Sang Yup Lee1,2,3*1Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, 2BioProcess Engineering Research Center, KAIST, 3BioInformatics Research Center, KAIST

Escherichia coli was metabolically engineered to produce a fumaric acid, an intermediate of the TCA cycle. To redirect carbon flux through the glyoxylate shunt, the iclR gene was deleted, and the fumA, fumB, and fumC genes were also deleted to enhance fumaric acid formation. The resulting strain was able to produce 1.45 g/L of fumaric acid in flask culture. Additionally, in silico flux response analysis was performed and the native ppc gene was overexpressed on plasmid level from tac promoter. This resulting strain produced 4.09 g/L of fumaric acid. Moreover, the arcA and ptsG genes were deleted to reinforce the oxidative TCA cycle, and the aspA gene was deleted to prevent the conversion of fumaric acid into L-aspartic acid. For enhanced glucose uptake rate and fumaric acid productivity, the native promoter of the galP gene was replaced with trc promoter and the lacI gene was also deleted to avoid the use of inducer. Fed-batch culture of the final strain CWF812 produced 28.2 g/L fumaric acid in 63 h with the overall yield and productivity of 0.389 g fumaric acid/g glucose and 0.448 g/L/h, respectively. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557).]

G003

Microbial Production of Gamma-butyrolactone via Metabolically Engineered Mannheimia succiniciproducens

Tong Un Chae1, Sol Choi1,2, and Sang Yup Lee1,2,3*1Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, 2BioProcess Engineering Research Center, KAIST, 3BioInformatics Research Center, KAIST

γ-Butyrolactone (GBL) is an important four carbon (C4) chemical, which has a wide range of industrial applications. GBL can be produced by acid treatment of 4-hydroxybutyric acid (4-HB), which is a derivative of succinic acid. Heterologous metabolic pathways were designed and established in succinic acid overproducing M. succiniciproducens LPK7 by the introduction of heterologous genes that encode succinyl-CoA synthetase, CoA-dependent succinate semialdehyde dehydrogenase, and either 4-hydroxybutyrate dehydrogenase in LPK7 (p3S4CD) or succinate semialdehyde reductase in LPK7 (p3SYCD). Fed-batch cultures of LPK7 (p3S4CD) and LPK7 (p3SYCD) resulted in the production of 6.37 and 6.34 g/L of 4-HB, respectively. Finally, GBL was produced by acid treatment of the 4-HB obtained from the fermentation broth. This study demonstrates that 4-HB, and potentially other four carbon platform chemicals, can be produced by the engineered rumen bacterium M. succiniciproducens. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557).]

G004

Production of Tyrosine and Cadaverine by Metabolically Engineered Escherichia coli Using Synthetic Small Regulatory RNA

Ji Yeon Ha1, Dokyun Na2, Seung Min Yoo1, and Sang Yup Lee1,3,4*1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), KAIST, 2School of Integrative Engineering, Chung-Ang University, 3Bioinformatics Research Center, KAIST, 4BioProcess Engineering Research Center, KAIST

Small regulatory RNAs (sRNAs) are gene expression regulators which act on post-transcriptional phase of bacterial gene expression. Here, we developed synthetic sRNA system to down-regulate the gene expression. The target mRNA binding region of MicC, one of the well-studied sRNAs in Escherichia coli, was replaced to translation initiation sequence of our target genes. We found that MicC scaffold based synthetic sRNA is properly repressed expression of DsRed2. Synthetic sRNAs was used for metabolic engineering to enhance the production of tyrosine and cadaverine. Through screening of 14 different strains which harboring synthetic sRNAs, we isolated a tyrosine producer producing 2 g/L of tyrosine. Using a library of 130 synthetic sRNAs, we screened knockdown targets that increase cadaverine productivity substantially. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557); the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) of the Ministry of Science, ICT & Future Planning through the National Research Foundation of Korea; the Commercializations Promotion Agency for R&D Outcomes (COMPA-2015K000365) funded by the Ministry of Science, ICT and Future Planning (MISP).]

Poster

www.msk.or.kr | 61

G005

Production of Phenol from Glucose by Metabolically Engineered Escherichia coli

Ji Yeon Ha1, Byoungjin Kim1, Hyegwon Park1, Dokyun Na2, and Sang Yup Lee1,3,4*1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), KAIST, 2School of Integrative Engineering, Chung-Ang University, 3Bioinformatics Research Center, KAIST, 4BioProcess Engineering Research Center, KAIST

Engineering E. coli for biological production of phenol is considered infeasible due to its toxicity and complex biosynthetic network. To address these challenges, we took advantage of the synthetic sRNA-based gene knockdown technology to simultaneously engineer 18 E. coli strains and screen them for higher phenol tolerance and precursor tyrosine availability. The knock-down of csrA and tyrR genes were identified to be crucial for redirecting carbon flux toward tyrosine and applied to all the E. coli strains with over-expression of the key genes for the biosynthesis of tyrosine and phenol (tyrosine phenol-lyase). The engineered strains showed substantially different metabolic phenotypes. The engineered BL21(DE3) strain produced 419 mg/L of phenol in flask culture and 1.69 g/L in fed-batch fermentation. A biphasic fed-batch fermentation using glycerol tributyrate was developed for in situ extraction while minimizing the phenol toxicity. The preferential partition of phenol from culture medium into glycerol tributyrate increased the titer and productivity to 3.79 g/L and 0.18 g/L/h, respectively. [This work was supported by the Intelligent Synthetic Biology Center through the Global Frontier Project (2011- 0031963) of the Ministry of Science, ICT & Future Planning through the National Research Foundation of Korea.]

G006

Rapid Multiple Gene Knockout System Using Integration-helper Plasmid

Ji Yeon Ha1, Chan Woo Song1, and Sang Yup Lee1,2,3*1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), KAIST, 2Bioinformatics Research Center, KAIST, 3BioProcess Engineering Research Center, KAIST

In this study, more rapid and efficient engineering method using integration helper plasmid in E. coli was developed. The integration helper plasmid, pCW611, has two recombinases which are expressed in reverse direction by two independent inducible systems. By using this system, required time and effort can be significantly reduced because the iterative transformation of the helper plasmid and curing steps are not required. We could delete one target gene in 3 days by using pCW611. To verify the usefulness of this gene manipulation system, the deletion experiments were performed for knocking out four target genes individually (adhE, sfcA, frdABCD, and ackA) and two genes simultaneously for two cases (adhE-aspA and sfcA-aspA). Also, fumaric acid producing E. coil strain was developed by deleting four target genes (fumB, iclR, fumA, and fumC) in 10 days as a proof-of-concept study.[This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557)]

G007

Native-sized Spider Silk Protein Production by Enhanced Glycine Pool

Ji Yeon Ha1, Xiao-Xia Xia1, Do Kyun Na2, and Sang Yup Lee1,3,4*1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), 2School of Integrative Engineering, Chung-Ang University, 3Bioinformatics Research Center, KAIST, 4BioProcess Engineering Research Center, KAIST

Naturally found spider silk and elastin protein show unique characteristics of highly repeated structure and extreme size as well as strength make them attractive to many industrial applications. However, exceptional structure and size limits expression in heterologous hosts, where the repetitive sequences in mRNA create extensive secondary structures. And these structures decrease ribosome processivity and assist mRNA degradation. Using the naturally found protein, spider dragline silk protein, we present techniques to solve biological problems that occurred: increasing the available ribosome pool and stabilizing mRNA for further degradation. Newly synthesized dragline silk protein produced increased titer than those reported previously, therefore proving that the strategies used were efficient. The results provide insight into approaches to control translation efficiency of proteins containing high molecular weight and highly repetitive sequence. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557).]

G008

Deletion of the Butyrate Kinase (buk) Gene is Essential for the High Butyric Acid Selectivity in Clostridium acetobutylicum

Seon Young Park1, Yu-Sin Jang1, and Sang Yup Lee1,2*1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute, 2BioProcess Engineering Research Center, Bioinformatics Research Center, Korea Advanced Institute of Science and Technololgy (KAIST)

An important industrial chemical, butyric acid, has been widely used in food, pharmacy, animal feed supplement, and chemical industries. Butyric acid producing Clostridium strains have a typical characteristic which is co-production of acetic acid with butyric acid. Thus, increasing the butyric acid selectivity is important for economical butyric acid production. However, increasing the butyric acid selectivity is difficult because of the complex metabolic pathways and physiologies of clostridia. In this work, Clostridium acetobutylicum was metabolically engineered to get the high butyric acid selectivity. The engineered C. acetobutylicum strain expressed the second butyrate kinase of C. acetobutylicum encoded by bukII gene instead of butyrate kinase I encoded by buk gene. Moreover, metabolic pathways were further engineered to enhance the NADH-driving force. In batch fermentation, the metabolically engineered C. acetobutylicum strain produced 32.5 g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AA ratio) of 31.3 g/g from 83.3 g/L of glucose at pH 6.0. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) and by the C1 Gas Refinery Project funded by the MSIP through the NRF of Korea (2015M3D3A1A01064918)]


62 | www.msk.or.kr

G009

Construction of Isopropanol-Butanol-Ethanol Production Platform in the Recombinant Clostridium Strains by Metabolic Engineering

Seon Young Park1, Joungmin Lee1, Yu-Sin Jang1, and Sang Yup Lee1,2*1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute, 2BioProcess Engineering Research Center, Bioinformatics Research Center, Korea Advanced Institute of Science and Technololgy (KAIST)

1-Butanol is an attractive biofuel which can replace gasoline with its specific properties. However, abnormal combustion can occur by its low octane rate. In this study, high amount of isopropanol-butanol-ethanol (IBE) mixture was produced in the recombinant Clostridium acetobutylicum strain by metabolic engineering. The hydGB-593 gene encoding putative ferredoxin:NADP+ reductase was co-expressed with the adhB-593 gene encoding NADPH-dependent primary/secondary alcohol dehydrogenase, and IBE titer was further improved. The final strain, a host strain for a large-scale fermentation, was used to prove its possibility for industrial IBE production. Although many clostridial strains contain the orthologous genes to hydGB-593 in their genome, C. acetobutylicum does not contain this gene. Our results showed that unlike other clostridial species, C. acetobutylicum might have a different mechanism of NADPH regeneration and the expression of hydGB-593 might increase the NADPH regeneration in C. acetobutylicum. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) and by the C1 Gas Refinery Project funded by the MSIP through the NRF of Korea (2015M3D3A1A01064918)]

G010

Metabolic Engineering of Escherichia coli for Short Chain Alkanes Production

Seon Young Park1, Yong Jun Choi1, and Sang Yup Lee1,2,3*1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 program), BioProcess Engineering Research Center, 2Center for Systems and Synthetic Biotechnology, Institute for the Biocentury, 3Department of Bio and Brain Engineering and Bioinformatics Research Center

So far, there have been many efforts on biofuel productions in microbes. However the production of highly demanded fuels like gasoline has not been achieved. In this research, Escherichia coli was developed for producing short-chain alkanes (gasoline), free fatty acids (FFAs), fatty esters and fatty alcohols. To prevent β-oxidation which degrades fatty acyl-CoAs generated in vivo, the fadE gene was deleted. For increasing the initiation of fatty acid biosynthesis, the fadR gene was knocked out and that consequently promoted the activity of FebH, 3-oxoacyl-ACP synthease and reduced the formation of unsaturated fatty acyl-ACPs. Additionally, for conversion of short chain fatty acyl-ACPs to the corresponding FFAs, a mutant TesA was conducted. The serial reactions of fatty acyl-CoA synthetase from E. coli, fatty acyl-CoA reductase from Clostridium acetobutylicum and fatty aldehyde decarbonylase from Arabidopsis thaliana converted the short chain FFAs into corresponding alkanes. Consequently, the final strain produced 580.8 mg/L of short chain alkanes. [This work was supported by the Advanced Biomass R&D Center(ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC-2011-0031350). Systems Metabolic Engineering work to Solve Climate Change was supported by Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1-A2A2026557).]

G011

Production of 1-Propanol by Metabolically Engineered L-threonine Overproducing Escherichia coli

Seon Young Park1, Yong Jun Choi1, Jin Hwan Park1, Tae Yong Kim1, and Sang Yup Lee1,2,3*1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 program), BioProcess Engineering Research Center, 2Center for Systems and Synthetic Biotechnology, Institute for the Biocentury, 3Department of Bio and Brain Engineering and Bioinformatics Research Center

1-Propanol is a greatly potential candidate of new biofuel which can substitute the gasoline. In this study, we introduced a novel pathway for 1-propanol biosynthesis by overproducing L-threonine in the Escherichia coli strain we have previously engineered. Formerly, Atsumi et al. produced 3.5 g/L of 1-propanol by expressing Methanococcus jannaschii citramalate synthase (CimA) which directly converts pyruvate into 2-ketobutyrate in metabolically engineered E. coli. The E. coli TH20 strain which overproduce L-threonine, developed in our previous study, was further engineered in this study. The flux response analysis for the carbon source usage optimization was conducted by in silico level, and then the knocking out of competing pathway was followed. The result strain succeeded in producing 1-propanol and the additional strain engineering further increased the titer. [This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC-2011-0031350). Systems Metabolic Engineering work to Solve Climate Change was supported by Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1-A2A2026557).]

G012

Production of Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by Metabolically Engineered Escherichia coli

Kyeong Rok Choi1, Jung Eun Yang1, Yong Jun Choi2, Seung Hwan Lee3, Bong Keun Song4, Si Jae Park5, and Sang Yup Lee1*1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus Program), BioProcess Engineering Research Center, KAIST, 2School of Environmental Engineering, University of Seoul, 3Dept. of Biotechnology and Bioengineering, Chonnam National University, 4Chemical Biotechnology Research Center, Korea Research Institute of Chemical Technology, 5Department of Environmental Engineering and Energy, Myongji University

Polyhydroxyalkanoates (PHAs), polyesters accumulated in many bacteria, has received great interest because of their unique characteristics, biode-gradable and biocompatible thermoplastic. Among various PHA copolymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] is an important copolymer because of its lower melting point and much better flexibility. So far, secondary carbon source was required to produce P(3HB-co-3HV). The toxicity of the auxiliary carbon source, however, retards cell growth while enhancing P(3HB-co-3HV) production. Here, we developed an E. coli stably synthesizing 3HB-CoA and 3HV-CoA in controlled ratio from glucose without feeding of exogenous auxiliary carbon source by metabolic engineering. Two different metabolic pathways for the production of propionyl-CoA from 2-ketobutyrate were constructed by introducing a series of heterologous genes into E. coli. The engineered strains were proven to efficiently synthesize P(3HB-co-3HV) independent of exogenous auxiliary carbon source. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557).]

Poster

www.msk.or.kr | 63

G013

Amino Acid L-arginine Production in a Metabolically Engineered Corynebacterium glutamicum

Kyeong Rok Choi1, Seok Hyun Park1, Hyun Uk Kim1,2, Tae Yong Kim1,2, Jun Seok Park3, Suok-su Kim3, and Sang Yup Lee1,2,4*1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus rogram), Center for Systems and Synthetic Biotechnology KAIST, 2BioInformatics Research Center, KAIST, 3Daesang Corporation Research Center, 4BioProcess Engineering Research Center, KAIST

Metabolic engineering approach was applied to Corynebacterium glutamicum for L-arginine production. To make C. glutamicum tolerant to L-arginine, random mutagenesis was first performed. Subsequently, arginine operon repressor proteins were inactivated, and the PPP flux was strengthened by downregulating pgi and overexpressing opcA, pgl, tal, tkt, and zwf. Then, Ncgl1221 encoding L-glutamate exporter was inactivated to channel L-glutamate to L-arginine formation. Additionally, argF and carAB expression levels were optimized for converting L-ornithine to L-citrulline effectively, followed by argGH operon overexpression. Fed-batch fermentation of the final strain was performed in a 1,500 L bioreactor resulting 81 g/L of L-arginine production. The approaches described here will be useful in developing corynebacteria regarding the production of arginine and its derivatives. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557).]

G014

Cadaverine Overproduction in a Metabolically Engineered Escherichia coli Cell Factory

Kyeong Rok Choi1, Zhi-Gang Qian1, Hye Min Park1, and Sang Yup Lee1,2,3*1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus rogram), Center for Systems and Synthetic Biotechnology KAIST, 2Institute for the BioCentury, KAIST, 3BioProcess Engineering Research Center, KAIST

A five-carbon diamine cadaverine can be used as a building block for sustainable polymer production. Bio-based production of cadaverine from renewable carbon sources is a promising and sustainable alternative to the current petrochemical production. Here, we report a series of metabolic engineering on Escherichia coli for the production of cadaverine from glucose in defined medium. To enrich cadaverine, degradative pathways on the target product were blocked. The engineered strain produced 9.61 g/L of cadaverine with a productivity of 0.32 g/L/h by fed-batch cultivation. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557).]

G015

L-Ornithine Bioproduction in Metabolically Engineered Corynebacterium glutamicum

Kyeong Rok Choi1, Seo Yun Kim1, Joungmin Lee1, and Sang Yup Lee1,2,3*1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus rogram), Center for Systems and Synthetic Biotechnology KAIST, 2BioInformatics Research Center, KAIST, 3BioProcess Engineering Research Center, KAIST

As a non-essential amino acid, L-ornithine has various applications in food industry. In this study, we report L-ornithine over-production by metabolically engineered Corynebacterium glutamicum ATCC 13032. To block competitive branch pathway and the conversion of L-ornithine to citrulline, proB and argF were first deleted, respectively. The L-arginine operon regulatory repressor gene argR was also removed to enhance the flux toward ornithine, resulting in 230 mg/L of L-ornithine from glucose in flask culture. Further engineering by plasmid-based overexpression of argCJBD genes from an arginine overproducer C. glutamicum ATCC 21831 resulted in the 7.19 g/L L-ornithine production. In effort to enrich the NADPH pool, the carbon flux was redirected towards the pentose phosphate pathway by changing the start codons of the pgi and zwf genes and replacing the native promoter of the tkt operon with the strong sod promoter. Fed-batch cultivation of the final strain YW06 (pSY223) achieved 51.5 g/L L-ornithine titer in 40 h with the overall productivity of 1.29 g/L/h. The results demonstrate the efficient L-ornithine production in metabolically engineered C. glutamicum. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557).]

G016

Engineering of Metabolic Flux into Novel Gamma-aminobutyric Acid Production Pathway by Introduction of Synthetic Scaffolds in Escherichia coli

Van Dung Pham, Sivachandiran Somasundaram, and Soon Ho Hong*

Department of Chemical Engineering, University of Ulsan

In general gamma-aminobutyric acid (GABA) pathway involves the decar-boxylation of glutamate, which is produced from sugar by Corynebacterium fermentation. GABA can be used for the production of pharmaceuticals and functional foods. Due to the increasing demand of GABA, it is essential to create an effective alternative pathway for the GABA production. In this study, Escherichia coli were engineered to produce GABA from glucose via GABA shunt, which consists of succinate dehydrogenase, succinate-semialdehyde dehydrogenase and GABA aminotransferase. The three enzymes were physically attached each other through synthetic scaffold, and the Krebs cycle flux was redirected to GABA pathway. By introduction of synthetic scaffold, 0.75 g/L of GABA was produced from 10 g/L of glucose at 30oC and pH 6.5. The inactivation of competing metabolic pathways provided 15.4% increase of the final GABA concentration. [This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant number: PJ011116) by RDA, and Basic Science Research Program by the Ministry of Education (NRF-2014R1A1A20-54726).]


64 | www.msk.or.kr

G017

Construction of Acidic Amino Acids Sensing Escherichia coli by Introduction of Chimeric Two-component System

Murali kannan Maruthamuthu and Soon Ho Hong*

Department of Chemical Engineering, University of Ulsan

In an attempt to create an acidic amino acid-sensing Escherichia coli, a chimeric sensor kinase (SK)-based biosensor was constructed using Pseudomonas putida AauS. AauS is a sensor kinase that ultimately controls expression of the aau gene through its cognate response regulator AauR, and is found only in P. putida KT2440. The AauZ chimera SK was constructed by integration of the sensing domain of AauS with the catalytic domain of EnvZ to control the expression of the ompC gene in response to acidic amino acids. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as the concentration of acidic amino acids increased. These data suggest that AauS-based recombinant E. coli can be used as a bacterial biosensor of acidic amino acids. By employing the chimeric SK strategy, various bacteria biosensors for use in the development of biochemical-producing recombinant microorganisms can be constructed. [This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant number: PJ011116) by RDA, and Basic Science Research Program by the Ministry of Education (NRF-2014R1A1A20-54726).]

G018

3D Wave of Fermented Soybean Extracts Suppress Proliferation of Human Breast Cancer MDA-MB-231 Cells

Jameon Park1,2 and Han Bok Kim1,2*1Department of Biotechnology, Hoseo University, 2The Research Institute for Basic Sciences, Hoseo University

3D wave part of fermented soybean extracts can be separated from matter of fermented soybean extracts. Fermented soybean extracts inhibit proliferation of human breast cancer MDA-MB-231 cells. In this study it is demonstrated that 3D wave of fermented soybean extracts could be transferred to water, and the water inhibited proliferation of human breast cancer MDA-MB-231 cells. Drug delivery into brain cells will be possible using 3D wave of its matter.

Keywords: 3D wave, fermented soybean, breast cancer

G019

Isolation of Biogenic Amine Non-producing Bacillus subtilis SCM121 and Medium Optimization for Improving Biomass by Response Surface Methodology

Su-Ji Jeong, Hee-Jong Yang, Seong-Yeop Jeong, Jeong Seon Eom, and Do-Youn Jeong*

Microbial Institute for Fermentation Industry (MIFI)

Biogenic amines are produced primarily by microorganisms found in fermented foods and are often implicated seriously poisoning in the body of human. Biogenic amines are thus considered a risk for human health. 620 strains were isolated from Korean traditional fermented food in Sunchang and investigated biochemical characterization and the ability of biogenic amines non-producing. SCM121 was selected based on the various properties such as extracellular enzyme, antioxidant and antimicrobial activities. The strain SCM121 was named as Bacillus subtilis SCM121 by 16S rRNA sequencing and biochemical characterization. And then, we investigated cell growth for application of industrial field, and optimized the culture medium constituents using response surface methodology. Plackett-Burman experimental design was used for screening of medium constituent, and sucrose, K2HPO4, and MgSO4 were selected as important factors for improving cell growth. In order to find out optimal concentration on selected constituents, we carried out central composite design. Consequently, optimal concentrations of sucrose, K2HPO4, and MgSO4 were predicted to be 3.5%, 0.8%, and 0.5%, respectively. By the model verification, we confirmed about 8-fold improvement of the dried cell weight from 1.1199±0.0306 g/L to 8.8778±0.081732 g/L when compared to basal medium.[Supported by grant from Cooperative Research Program for Agriculture Science & Technology Development (No. PJ009990032014).]

G020

Isolation of Bacillus subtilis SCM146 Having Antimicrobial Activity and Medium Optimization for Improving Biomass by Response Surface Methodology

Hee-Jong Yang, Su-Ji Jeong, Seong-Yeop Jeong, Jeong Seon Eom, and Do-Youn Jeong*


620 strains were isolated from Korean traditional fermented food in Sunchang and investigated biochemical characterization and the ability of biogenic amines non-producing. SCM146 was selected based on the various properties such as extracellular enzyme, antioxidant activities, and et al., and was named as B. subtilis SCM146 by 16S rRNA sequencing and biochemical characterization. And then, we investigated cell growth for application of industrial field, and optimized the culture medium constituents using response surface methodology. Plackett-Burman experimental design was used for screening of medium constituent, and sucrose, yeast extract, tryptone, and K2HPO4 were selected as important factors for improving cell growth. In order to find out optimal concentration on selected constituents, we carried out central composite design. Consequently, optimal concentrations of sucrose, yeast extract, tryptone, and K2HPO4 were predicted to be 3.5%, 3.5%, 3.5%, and 1.1%, respectively. By the model verification, we confirmed about 7.5-fold improvement of the dried cell weight from 0.725±0.024 g/L to 5.5333±0.0701 g/L when compared to basal medium. Finally, we carried out experiments for establishing optimal pH and culture temperature, confirmed maximal cell growth at 30℃ and initial pH 7.0.[Supported by grant from Cooperative Research Program for Agriculture Science & Technology Development (No. PJ009990032014).]

Poster

www.msk.or.kr | 65

G021

Isolation of Biogenic Amine Non-producing Lactobacillus brevis SCML67 and Medium Optimization for Improving Biomass Using Statistical Technique

Su-Ji Jeong, Hee-Jong Yang, Seong-Yeop Jeong, Jeong Seon Eom, and Do-Youn Jeong*


583 strains were isolated from Korean traditional fermented food in Sunchang and investigated biochemical characterization and the ability of biogenic amines non-producing. SCML67 was selected based on the various properties such as extracellular enzyme, antioxidant activities, and ability of biogenic amine non-producing, and named as L. brevis SCML67 by 16S rRNA sequencing. And then, we investigated cell growth for application of industrial field, and optimized the culture medium constituents using response surface methodology. Plackett-Burman experimental design was used for screening of medium constituent, and molasses, CSP, and urea were selected as important factors for improving cell growth. In order to find out optimal concentration on selected constituents, we carried out central composite design. Consequently, optimal concentrations of molasses, CSP, and urea were predicted to be 1.5%, 2.0%, and 1.0%, respectively. By the model verification, we confirmed about 1.3-fold improvement of the dried cell weight from 2.457±0.006 g/L to 3.1088±0.0338 g/L when compared to basal medium. Finally, we carried out experiments for establishing optimal pH and culture temperature, confirmed maximal cell growth 4.12±0.0985 g/L at 37°C and initial pH 7.0.[Supported by High value-added Food Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries (No. 313037-3).]

G022

Isolation of Biogenic Amine Non-producing Lactobacillus brevis SCML458 Having Antioxidant Activity and Medium Optimization for Improving Biomass

Hee-Jong Yang, Su-Ji Jeong, Seong-Yeop Jeong, Jeong Seon Eom, and Do-Youn Jeong*


Antioxidant are substances that may protect cells from the damage caused by unstable molecules known as free radicals. Free radical damage may lead to cancer. In this study, 583 strains were isolated from Korean traditional fermented food in Sunchang and investigated biochemical characterization. SCML458 was selected based on antioxidant activities and ability of biogenic amine non-producing, and named as L. brevis SCML458 by 16S rRNA sequencing. And then, we investigated cell growth for application of industrial field, and optimized the culture medium constituents using response surface methodology. Plackett-Burman experimental design was used for screening of medium constituent, and protease peptone, CSP, and yeast extract were selected as important factors for improving cell growth. In order to find out optimal concentration on selected constituents, we carried out central composite design. Optimal concentrations of protease peptone, CSP, and yeast extract were predicted to be 2.0%, 2.5%, and 2.0%, respectively. By the model verification, we confirmed about 1.3-fold improvement of the dried cell weight from 2.40±0.01 g/L to 2.97±0.05 g/L when compared to basal medium. Finally, we carried out experiments for establishing culture conditions, and confirmed maximal cell growth 6.25±0.02 g/L at 37°C and initial pH 8.0.[Supported by High value-added Food Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries (No. 313037-3).]

G023

KCl Effect on Growth of Leuconostoc mesenteroides CS-5 under the High Salt Concentration

Oh Sik Kwon

Major in Biological Science, College of Natural Science, Keimyung University

Characteristics of Leuconostoc mesenteroides strains isolated from chonggak kimchi was studied to identify salinity nature. Among the isolates, a strain named as CS-5 showed an optimal growth temperature that ranged from 24°C to 28°C. Interestingly the CS-5 was turned out to be a dextran producing strain. It could grow in the media containing 6% NaCl. At 5% salinity the CS-5 revealed much better growth in media containing glucose and fructose as a monosaccharide than a strain of CL-1 that could not grow at all. From tests of disaccharides (maltose, sucrose), the CS-5 showed strong growth nature at 6% salinity in which the CL-1 could not grow at all. This kind of salt tolerance was also appeared in the media containing trehalose, melibiose and cellobiose that were known to be hard to ferment. Sole addition of KCl (40 mM, 54 mM) on growth media for CS-5 showed small increase for its cell growth determined by pH of supernatant from fermented media. However, addition of 40 mM KCl in media containing 4% to 8% NaCl enhanced growth of CS-5 dramatically. This implies that higher salt concentration can be overcome by addition of KCl during cell growth of CS-5 as lactic acid bacteria. From a test for dextran production, the CS-5 showed higher values of viscosity (0.153 Pa.sn) in media containing 30% sucrose (w/v) comparing to the controls (0.01 Pa.sn).

Keywords: Dextran, Chonggak Kimchi, Leuconostoc mesenteroides, Salinity

G024

Oxidation of Methane throuth Component Interactions from Type II Methanotrophs

Min Young Song1, Eun Taek Seol1, and Seung Jae Lee1,2*1Department of Chemistry, Chonbuk National University2Research Institute of Physics and Chemistry, Chonbuk National University

Methane hydroxylation through methane monooxygenases (MMOs) is a key aspect due to their control of the carbon cycle in the ecology system and recent applications of methane gas in the field of bioenergy and bioremediation. Methanotropic bacteria perform a specific microbial conversion from methane, one of the most stable carbon compounds, to methanol through elaborate mechanisms. MMOs express particulate methane monooxygenase (pMMO) in most strains and soluble methane monooxygenase (sMMO) under copper-limited conditions. The mechanisms of MMO have been widely studied from sMMO included in the bacterial multicomponent monooxygenase (BMM) superfamily. Mechanism studies of sMMO have been intensively studied by the supports of advanced biophysics, especially in the Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b strains. Structural studies of three components of sMMO, a hydroxylase (MMOH), a regulatory component (MMOB), and a reductase (MMOR), have provided crucial information about their catalytic activities. In this study, we report successful growing and expression from type II methanotrophs, Methylosinus sporium and Methylocystis sp. Strain M.[Supported by grant from KRF of Korea.]


66 | www.msk.or.kr

G025

Stability and Inactivation Mechanism of Porcine Parvovirus against High Temperature Treatment

Sang Eun Han1, Jung Eun Bae2, and In Seop Kim1*1Department of Biological Sciences and Biotechnology, Hannam University2BioPS

Viral contamination of human plasma or mammalian cell cultures in GMP manufacturing facility represents a serious safety threat to biopharmaceutical industry. Such adverse events usually require facility shutdown for cleaning/ decontamination, and thus result in significant loss of production and/or delay of product development. Therefore, treatment with steam and/or dilute NaOH are commonly used to disinfect manufacturing vessels and tools in the biopharmaceutical industry. Porcine parvovirus (PPV) has been known to show the highest resistance to a range of physico-chemical treatment. PPV is the best model virus for cleaning validation. In this study, we examined inactivation kinetics of PPV against high temperature treatments (70, 80, 90°C) in order to optimize the thermal treatment process to inactivate viral contaminants. PPV showed resistance against 90 min treatment at 70°C and 80°C with the log reduction value of 1.44 and 3.91, respectively. PPV was completely inactivated after 20 min treatment at 90°C. Thermal inactivation mechanism was investigated by quantitative real-time PCR determination of leakage of DNA using endonuclease treatment to thermally inactivated PPV as well as intact PPV. From this study, it was found that thermal inactivation at 90°C was not due to disintegration of the viral capsid. Further study to elucidate the thermal inactivation mechanism of PPV is currently under way.

G026

Biofunctionality and Bioconversion Activities of KA111 (Pseudomonas fragi), KA115 (Bacillus methylotrophicu), KA182 (Bacillus subtilis), KA198 (Bacillus safensis), KA217 (Bacillus pumilus), KA222 (Bacillus licheniformis)

Sung-Ho Joo, Won Mun Kim, Kwang-Su Lee, and Ki-Sung Lee*

Department of Biology & Medicinal Sciences, Pai Chai University

In this study, we conducted the studies on the biofunctionalities and bioconversion activities of the six strains. In order to find out new functional resources and to elucidate new biofunctionalities and bioconversion activities of six kinds of microorganisms, we performed the following experiments. These biofunctional resources are involved in physio-biochemically important amino acids such as ornithine (Orn), citrulline (Cit) and r-aminobutyric acid (GABA). We isolated these strains (KA111, KA115, KA182, KA198, KA217, and KA222), which were tested and analyzed bioconversion activities transforming Arg (arginine) into Orn and Cit. As the results, all of the six strains showed bioconversion activities converting Arg into Orn and Cit directly or indirectly.Also, their bioconversion activities showed very strong regardless of complex medium, malt extract medium composed of natural product, but those showed weak in the minimal medium. All of the six strains could bioconvert Arg into Orn and/or Cit almost 100% efficiently not only on the complex medium but also on MEM. On the other hand, in the case of on the minimal medium, five strains (KA115, KA182, KA198, KA217, and KA222) could bioconvert Arg into Orn more than 50%, but KA111 strain couldn’t bioconvert. [This work was supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education and National Research Foundation of Korea (2015035949).]

G027

Bioconversion Efficiency in Accordance with the Substrate Concentrations

Sung-Ho Joo, Won Mun Kim, Kwang-Su Lee, and Ki-Sung Lee*

Department of Biology & Medicinal Sciences, Pai Chai University

We carried out experiments related with bioconversion efficiency according to the higher concentrations of substrate (Arg;Arginine). Of the biofunctional resources, emphases are placed on physio-biochemically important amino acids such as ornithine (Orn), citrulline (Cit) and r-aminobutyric acid (GABA). We isolated six strains (KA111, KA115, KA182, KA198, KA217, and KA222), which were tested and analyzed bioconversion activities under changing the concentrations of Arg at the higher levels of 3 times (3X) and 5 times (5X). As the results, when we applied high concentration of Arg with 3 times (3X) or 5times (5X) that is higher than the normal condition (control group in the media), KA182, KA198, KA217, and KA222 did convert Arg into Orn and/or Cit more than amounting to 70% in complex medium. But in the minimal medium only KA222 strain did convert Arg into Orn and/or Cit. On the other hand, KA111, KA115 did not covert. Finally, multi-biofunctional microbes (KA111, KA115, KA182, KA198, KA217, and KA222) were related to biodegradability and bioconversion activity. According to our results, we suggest that our identified bio-functional microbes had effect on bio-ethanol process, bioremediation and regeneration of polluted waste materials.[This work was supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education and National Research Foundation of Korea (2015035949).]

G028

Characterization and Validation of the Modified tuf Promoter to Develop the Efficienct Recombinant Protein Expression Vector System for Lactococcus lactis IL1403

Inseon Kim1,2, Jinduck Bok2, Sangkee Kang2, Chongsu Cho1, and Yunjaie Choi1*1Department of Agricultural biotechnology, Seoul National University2Institute of Green-Bio Science & Technology, Seoul National University

Lactococcus lactis (L. lactis) has been known to be a good recombinant host for expression of recombinant proteins with ease of manipulation and a live oral vaccine carrier. Therefore, many approaches have been attempted to improve the recombinant protein in L. lactis. To improve the level of protein expression in this study, we developed strong promoters for L. lactis by modifying the tuf promoter (189 bp) which was previously studied by our group. The tuf promoter was modified by repeating core region (119 bp) such as -35 and -10 cis-acting elements to recruit the transcriptional initiation factors and altering the ‘G’, ‘C’ sequences to the ‘A’ at downstream of ribosome binding site. We successfully constructed mtuf1, mtuf2, mtuf3, mtuf1-1, and mtuf2-1 promoters without superfluous sequences by serial PCR steps. For validation of the efficacy of modified promoters, we carried out luciferase assay, qRT-PCR, western blot, and in vivo imaging. We have found that mtuf1, mtuf2, and derivative mutf2-1 promoters expressed higher level of protein than original tuf promoter. In specific, mtuf2-1 has shown great potential for application in construction of recombinant L. lactis as oral delivery vehicle. [Supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA)(115084-2)]

Poster

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G029

Inhibition of Enterotoxigenic Escherichia coli Cell-growth by ETEC Specific Binding DNA Aptamer

Woo-Ri Shin1, Sang-Hee Lee1, Ji-Young Ahn1, Jiho Min2, and Yang-Hoon Kim1*1Department of Microbiology, College of Natural Sciences, Chungbuk National University, 2Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University

Enterotoxigenic Escherichia coli (ETEC) strains may cause diarrheal diseases in humans and farming animals. For pigs and calves, K88 and K99 fimbriae, which are expressed by ETEC strains, are responsible for most neonatal infections and the majority of diarrheal infections. Aptamer is single-stranded DNA or RNA that can bind to various targets with high specificity. It has a number of advantages like high stability at high temperature and pH easily modified with various functional groups. In this study, we generated DNA aptamers specific to ETEC (K88, K99 strains) using a whole bacterial Cell-SELEX process and characterized their affinity, and specificity to other cells. These aptamers modulate processed bacterial cell growth rate in ETEC K88 & K99. [This work was carried out with the support of "Cooperative Research Program for Agriculture Science & Technology Development (Project title: Development of Monitoring and Diagnostic Method for Environmental Animal Disease, Project No: PJ010530)" Rural Development Administration, Republic of Korea.]

G030

Human Immunodeficiency Virus Type 1 Safety of Blood Coagulation-related Plasma Products

Jung Eun Bae, Eun Kyo Jeong, Dong Joo Yu, Da Jeong Kim, Jung Sun Jeong, Sang Eun Han, and In Seop Kim*

Department of Biological Sciences and Biotechnology and Center for Biopharmaceuticals Safety Validation, Hannam University

The production of plasma derivatives of human origin must take into account the possible presence of pathogenic viruses in the original material. Thus, special precaution must be taken during the production to assure against the possibility of the products transmitting infectious disease to the recipients. The most dangerous blood-borne virus of clinical concern is human immunodeficiency viruses type I (HIV-1). However, there has been limited information about HIV safety for fibrin glue, antithrombin III, and factor IX. This study was thus designed to evaluate the HIV-1 inactivation efficacy of manufacturing processes for fibrin glue, antithrombin III, and factor IX. Solvent/detergent (S/D) and 60℃ heat treatments during the manufacture of fibrin (fibrin glue syringe I) have sufficient HIV-1 inactivation efficacy with log reductions values (LRV) of ≧3.03 and ≧4.07, respectively. S/D and nano-filtration processes during the manufacture of thrombin (fibrin glue syringe II) have sufficient HIV-1 inactivation and removal efficacy with LRV of ≧3.04 and ≧4.82, respectively. Faction II+III precipitation, 60°C heat treatment, and nano-filtration processes during the manufacture of antithrombin III have sufficient HIV-1 inactivation and removal efficacy with LRV of 3.85, ≧3.84 and ≧4.17, respectively. S/D and freeze-drying processes during the manufacture of factor IX have sufficient HIV-1 inactivation efficacy with LRV of ≧3.35 and ≧3.69, respectively.

G031

CTHRC1 Protein Binding RNA-aptamer Selection by SELEX

Hee-Young Cho1, Woo-Ri Shin1, Kyeong-Ah Lee1, Se Hee Lee1, Simranjeet Singh Sekhon1, Ji-Young Ahn1, Dae-Ghon Kim2, Jiho Min3, and Yang-Hoon Kim1*1Department of Microbiology, College of Natural Sciences, Chungbuk National University, 2Research Institute of Clinical Medicine of Chonbuk National University, 3Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University

Hepatocellular carcinoma (HCC) is the most frequent type of liver cancer. Alpha-fetoprotein (AFP) is a popular biomarker for HCC, however AFP is not detected in first stage. Collagen Triple Helix Repeat Containing 1 (CTHRC1) protein is overexpressed in HCC, which acceleration tumor cell invasion. The reason we use aptamer to detect CTHRC1 protein is aptamer’s many advantages. Aptamer have less sensitive to high temperature, various application and high affinity to their target. Generated RNA aptamer candidates were using Systematic Evolution of Ligands by EXponential enrichment (SELEX) process. 32 RNA aptamer candidates were selected by real-time PCR and T-vector cloning. Followed by Surface Plasmon Resonance (SPR) process to obtain high affinity aptamer which is specific binding to CTHRC1 protein. These aptamers can be treated in human Hepatocellular carcinoma cell lines. The objective of our research to reduce the sizes of the cancer cells by aptamer treatment. [This study was supported by Fund of Biomedical Research Institute, Chonbuk National University Hospital (20120801002). This work was supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education and National Research Foundation of Korea (NRF-2015H1C1A1035921).]

G032

Isolation and Identification of Strain for Saccharina japonica Fermentation

Hyeon Song Oh and Youn Tae Chi*


Saccharina japonica is mainly distributed in the coast of East Asia, including Korea, China and Japan. Saccharina japonica contains large amounts of functional material, such as alginate and fucoidan. Also it includes various inorganic salts such as calcium, magnesium and iodine. The main component of Saccharina japonica, alginate, has several functions such as obesity inhibition, constipation relief, anti-cholesterol effect, detoxification and heavy metal emission. In addition, α-D-mannuronic acid and β-L-guluronic acid formed from depolymerization of alginate are effective in anti-cancer and anti-bacterial actions. However, breakdown and absorption of alginate do not occur efficiently in the human body. Fermentation can be one of the solution for this limitation. Therefore, in this study, we aimed to find the best strains for fermenting Saccharina japonica.


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G033

Effect of Before and After Fermented Dendropanax morbifera Extract on HEK293 and PANC-1 Cells

Ra Min Seo and Youn Tae Chi*


Dendropanax morbifera is a genus of flowering plants in the family Araliaceae. Today, it attracts people’s attention for positive effects on human health so that many studies have been carried out and are still ongoing. Several studies found extracts from Dendropanax morbifera have enormous implications including anti-cancer and anti-bacterial activity. However, there is limitation that all these beneficial compounds the plant has are not assimilated into the human body. Fermentation is suggested as a possible solution to overcome the restriction on absorption. Thus, this study is implemented to examine, to what extent, absorption of the plant’s advantageous compounds is achieved by using fermented Dendropanax morbifera compared to its extract. To do so, HEK293 and PANC-1 cells were used for MTT assay.

G034

Comparing the Ingredients and Antioxidant Effects of Dendropanax morbifera Fermentation Using Microorganism

Ho Min Song, Sang Hoon Na, and Youn Tae Chi*


Dendropanax morbifera is native to southern Korea its sap is used as paint of traditional crafting, and now in the spotlight as a material for functional foods. Recent studies reported Dendropanax morbifera extract shows anti-bacterial, anti-cancer, antioxidant excellent effects. In this study, Dendropanax morbifera extract is the fermentation using microorganisms to make sure they are easier absorption by body and compare its antioxidant effects and the end of fermentation ingredients to before fermented.

G035

Isolation and Identification of Ground Microorganism to Find New Beneficial Microbial Agents

Jin Sung Kim and Youn Tae Chi*


We isolated and identified micro organism from ground sample of Chonnam area to find new beneficial microbial agents. We characterized the identified new micro organisms and examined their anti-microbial activity for plant disease. In addition, antibiotics extracted from the micro organisms was tested to find out the best condition for production. Through this study, we found that the identified ground micro organisms can be used for potential beneficial microbial agents.

G036

Screening and Application of Xylanase Producing Sphigobacterium sp. A10 Strain

Sang Hoon Na and Youn Tae Chi*


The xylan is a group of hemicelluloses that are found in plant cell wall and some algae. Xylan are polysaccharides made from units of xylose. So, xylanolytic enzymes from microorganism have attracted a great deal of attention because of their biotechnological characteristics in various industrial processes, related to pulp, food, feed, ethanol, and paper industries. A microbial screening of xylanase producer was carried out in this study. About 250 bacterial strains were isolated from soil sample. Among these isolated bacteria, a isolate A10 as good xylanase producers were identified. The bacterial strain A10 identified as Sphigobacterium sp. A10, was cultivated on submerged fermentation using as substrate xylan, pectin, mannan, lignin, polygalcacturonic acid and hemp stalks extract. Hemp stalks extract show a good xylanase activity after 48 hour of fermentation. Sphigobacterium sp. A10 producing xylanase was showed optimum pH and tempertature for enzyme activity at 10, and 53 degree C, respectively.

Poster

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G037

Dyeing Properties and Antibacterial Activity of Natural Pigment from Marine Bacteria



In this study, dyeability on mutifiber fabrics and antibacterial activities using natural pigment derived from marine bacteria were investigated. The violet colourant was produced by cultivation of Microbulbifer sp. PPB 12 using marine broth 2216 for 3 days. The violet pigment dissolved in 20% ethanol could be dyed on bleached cotton, diacetate, and especially polyamid. Optimum temperate, time, pH and bath ratio under dyeing condition were 80–90°C, more than 1 hour, pH 4–6 and 1:25, respectively. Mordanting treatment after dyeing 10 minutes was suited to color expression, but no significant difference was showed from being not treated. Also, the violet pigment showed antibacterial activity against Bacillus subtilis. As a results, the natural colourant from marine bacteria is considered to be utilized for manufacturing polyamide garment with antibacterial activity.[Supported by Ministry of Education (1345231167)]

G038

The Physicochemical Stabilities of Pigment Extracts from Erythrobacter sp. PPB2-8



We investigated the physicochemical stabilities and antibacterial activitiy of ethanol- extracted pigment from marine bacterium Erythrobacter sp. PPB2-8. The bacterial pigment of strain PPB2-8 was very stable at pH 8–9 and -20~40°C. In the presence of light, the pigment was also stable, showing more than 77% of remaining absorbance during 15 days at 25°C. The stability of the pigment, when metal ions were present, was higher stability in all examined metal ions except for Cu2+ and Al3+, especially in the presence of Na+. The result indicates that the bacterial pigments from marine bacterium, Erythrobacter sp. PPB2-8 showed higher physicochemical stability. Therefore, the results suggest that these bacterial pigments could be used as a natural colorant.[Supported by Ministry of Education (1345231167)]

G039

Development of Home-made Gibson Assembly Enzymes for Do It Yourself Biologists

Jaewon Kim, Saeyoung Lee, Hongjae Park, Kyoungwoo Jang, and In-Geol Choi*

Department of Biotechnology, Korea Univeristy

Gibson assembly (GA) is a popular cloning method used in the joining of multiple DNA fragments in a test tube. Using the commercial kit costs more than $10 per reaction and still demands good hands in unexperienced labs. In order to provide a home-made GA kit for ‘Do It Yourself’ (DIY) biologists, we sought candidate enzymes for the home-made kit. We expressed three enzymes under a T7 expression system: a thermophilic 3’ to 5’ proofreading DNA polymerase from Pyrococus furiosus (PfuPol), a thermophilic NAD+ dependent DNA ligase from Auqifex pyrophilus (ApyLig) and a DNA exonuclease from T7 bacteriophage (T7Exo). The activity of each enzyme was optimized for various reaction conditions. By mixing three enzymes in a combinatorial way, we built a home-made GA kit for DNA cloning in an isothermal reaction and determined the optimal condition. When we tested with two DNA molecules (200 ng each) overlapped 40 bps in the final reaction mixture of 15 ul, the optimal temperature and reaction was 65°C for 15 min. The best result was obtained at the molar ratio of 1:15:50 of T7Exo, PfuPol, and ApyLig, respectively. We propose this home-made GA kit as a basic cloning tool for DIY biologists as well as a gene assembly tool for a poor laboratory aiming synthetic biology technology.

G040

RapB Controls Cell Adhesion and Migration in Dictyostelium

Byeonggyu Park and Taeck J. Jeon*

Department of Life Science & BK21 – Plus Research Team for Bioactive Control Technology, College of Natural Sciences, Chosun University

Ras proteins are small, monomeric GTPases that act as crucial regulators of a number of cellular signaling pathways, including proliferation, cell migration, differentiation and apoptosis. In Dictyostelium genome database, it has been reported that there are 19 Ras subfamily proteins. The functions of most of these Ras proteins in development and cell migration have not been studied yet. Here we investigated the roles of RapB in cell migration and development. RapB is a Ras subfamily protein showing the highest homology (48.9% identity in amino acid sequences) with RapA, which is a key regulator in cell adhesion and cell migration. Similar to RapA-overexpressing cells, cells expressing constitutively active form of RapB displayed flattened and spread morphology and strong cell-substratum adhesion strength compared to wild-type cells. In addition, RapB-CA cells showed slower migration speed and migration indexes than wild-type cells in electrotaxis, and slightly delayed development. GFP-fusion RapB appears to localize to the cell cortex. These results suggest that RapB plays some important roles in cell adhesion and cell migration, similar to RapA.[Supported by Basic Science Reserach program through NRF]


70 | www.msk.or.kr

G041

Cytokinesis Defects of rapGAP9 null Cells are Rescued by Expressing Truncated GAP-Domain Proteins

ARa Lee and Taeck J. Jeon*

Department of Life Science & BK21-Plus Research Team for Bioactive Control Technology, College of Natural Sciences, Chosun University

Rap1 is an important regulator in cell adhesion and cell migration. It has recently been reported that spatial and temporal regulation of Rap1 activity by RapGAPs is required for development and cell migration in Dictyostelium. RapGAP1 is involved in spatiotemporal regulation of cell adhesion at the front of chemotaxing cells. RapGAP3 is associated with morphogenesis and development. RapGAP9 is involved in cell adhesion and cytokinesis. Here, we investigated overlapping/specific functions of RapGAP1, RapGAP3, and RapGAP9 in morphogenesis, adhesion, cytokinesis, and development by analyzing phenotypes of rapGAP null cells expressing each RapGAP protein. Interestingly, almost all phenotypes of rapGAP1, rapGAP3, and rapGAP9 null cells in development and cytokinesis were complemented by expressing any one of the RapGAP proteins (RapGAP1, RapGAP3, and RapGAP9), suggesting that the functions of these RapGAP proteins are highly overlapped. It has been known that rapGAP9 null cells are highly multinucleated. These cytokinesis defects of rapGAP9 null cells were completely by expressing full-length RapGAP9 or truncated proteins containing the GAP domain. In contrast rapGAP9 null cells expressing truncated proteins without the GAP domain showed no rescued phenotypes in cell adhesion, cytokinesis and development. These results suggested that the GAP domain is essential for the RapGAP9’s functioning in Dictyostelium cells.[Supported by Base Science Research Program through NRF]

G042

Proper Regulation of CBP7, a Calcium Binding Protein, is Required for Development in Dictyostelium

Dong-Yeop Shin and Taeck J. Jeon*


Calcium and calcium binding proteins are necessary for diverse intracellular signaling. In Dictysotelium, fourteen genes encoding calcium binding proteins (CBP) have been found. The functions of most of these CBP proteins in development have not been studied yet. CBP7, one of 14 CBPs, is composed of 169 amino acids and contains four EF-hand domains. Here, we investigated roles of CBP7 in Dictyostelium development and found that CBP7 appears to play some important roles in the cell aggregation step during development. Surprisingly, cells expressing CBP7 showed severe development defects, complete loss of development, while wild-type cells formed aggregates within 6-8hr development and finally fruiting bodies. CBP7-overexpressing cells did not even aggregate and just stayed at the single-cell growing stages in the development-inducing condition. Cell sorting experiments using mixed cells of wild-type cells and CBP7 overexpressing cells confirmed that CBP7-overexpressing cells had much lower migration and aggregation speed in the aggregation stage of development compared to wild-type cells. The third one of four EF-hand domains in CBP7 appear to be essential for functions of CBP7 in development. Cells expressing CBP7 with mutations in the third EF domain showed normal development. Our results suggest that CBP7 plays an important role in development through the third EF-hand domain, possibly calcium binding. [Supported by Basic science Research program through NRF]

G043

Adaptive Engineering of a Hyperthermophilic Archaeon on CO and Discovering the Underlying Mechanism by Multi-omics Analysis

Seong Hyuk Lee1,2, Min-Sik Kim3, Jae-Hak Lee1, Tae Wan Kim1,2, Seung Seob Bae1, Sung-Mok Lee1, Hae Chang Jung1,2, Tae-Jun Yang1, Ae Ran Choi1, Yong-Jun Cho4, Jung-Hyun Lee1,2, Kae Kyoung Kwon1,2, Hyun Sook Lee1,2, and Sung Gyun Kang1,2*1Korea Institute of Ocean Science and Technology, 2Department of Marine Biotechnology, Korea University of Science and Technology, 3Korea Institute of Energy Research, 4Chunlab, Inc.

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.[This work was supported by the KIOST in-house program (PE99413), the Development of Biohydrogen Production Technology Using the Hyperthermophilic Archaea program of the Ministry of Oceans and Fisheries in the Republic of Korea, and the Korea C1 Gas Refinery R&D Center program of the Ministry of Science, ICT and Future Planning in the Republic of Korea.]

G044

Adaptive Evolution of a Hyperthermophilic Archaeon Thermococcus onnurineus NA1 during Growth on Formate

Hae-Chang Jung1,2, Seong Hyuk Lee1,2, Jung-Hyun Lee1,2, Hyun Sook Lee1,2, and Sung Gyun Kang1,2*1Korea Institute of Ocean Science and Technology2Department of Marine Biotechnology, Korea University of Science and Technology

The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been reported to grow on formate coupled with H2 production and ATP generation. To develop the strain for the enhanced H2 production on formate, adaptive evolution was adopted by transfering T. onnurineus NA1 to formate containing medium. Through serial transfers of batch cultures, physiological changes were monitored and gradual increases in cell density, H2 production rate and formate consumption rate were observed. After 156 transfers, the evolved strain showed 1.4-, 1.3- and 1.3-fold higher values in maximum cell density, H2 production rate and formate consumption rate, respectively, in comparison with those of parental strain. In order to obtain an integrative understanding of the genetic and phenotypic changes during evolution on formate, genome sequencing was performed. As a result, we discovered eleven mutation sites, including 2 insertions, 2 deletions and 7 substitutions either at the 2 intergenic or 9 coding regions. Further mutational studies would be required to assess the contribution of gene mutations to the phenotypic changes during evolution. The potential of the engineered strains as a H2 producer will also be investigated by kinetic analysis in a bioreactor. [This work was supported by the KIOST in-house program (PE99413) the Development of Biohydrogen Production Technology Using the Hyperthermophilic Archaea program of the Ministry of Oceans and Fisheries in the Republic of Korea.]

Poster

www.msk.or.kr | 71

G045

DReS: A Direct Cloning Technique Using Bacteriophage Recombination Systems

Kyoungwoo Jang, Linh Thuy Do, Hongjae Park, and In-Geol Choi*

Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University

Molecular cloning in conventional genetic engineering has been dependent on various molecular biology enzymes such as restriction enzymes and ligases. Synthetic biology employs novel cloning techniques such as CPEC, LIC, SLICE or Gibson assembly. Recombineering (recombination-mediated genetic engineering) is a synbio cloning method using homologous recom-bination. This method not only allows successive joining of DNA fragments having overlaps but also manipulates very large DNA fragments such as bacterial artificial chromosomes or chromosomes. In order to clone or disrupt a genomic region of bacteria, we developed a recombieering method based on bacteriophage recombinases designated as DReS (Dual Recombination System) using both λ (Red α/β/γ) and Rac prophage recombination system (RecET). The method is mainly focused on ‘circular-circular recombination (CCHR)’. Using the CCHR, we attempted a direct cloning of a large chromosomal region into a plasmid vector or deletion of genomic regions in bacteria. In comparison to known recombinations systems (λ-Red and RecET), DReS was most efficient for CCHR. The successful recombination rate of DReS was 2.4-fold and 8.7-fold higher than those of RecET and λ-Red systems, respectively. Although the optimization of recombination is required for various conditions (e.g. hosts, vectors, promoters, etc.) , DReS has a potential for assembly of a large size of synthetic DNA or cloning of a whole metabolic pathway in a single plasmid.

G046

A Bacterial Cell-based Biosensor Using Bacterial-two Hybrid System for the Detection of Endocrine Disrupting Chemicals

Su Hyun Ryu, Yoon Mo Yang, Yeh Eun Lee, and Jin Won Lee*

Department of Life Science and Research Institute for Natural Sciences, Hanyang University

Endocrine disrupting chemicals (EDCs) such as bisphenol A and nonylphenol are organic pollutants which can interfere with the endocrine system. Estrogen receptor (ER) is a hormone receptor which binds 17β-estradiol during a normal response. However, ER can also bind to EDCs which interfere with the activity of ER. Thus, ER itself can be used as sensor for the detection of EDCs. Here we report a new type of bacterial cell-based EDC-sensing system. Our sensor system utilizes bacterial two hybrid system composed of ligand-dependent interaction between human ERα ligand binding domain (hERα LBD) and hERα coactivator, RIP140. We constructed two fusion genes: RNA polymerase αNTD-hERα LBD and λCI repressor- RIP140. And these fusion genes were introduced into the E. coli strain having λ operator-lacZ reproter fusion gene. In the presence of nanomolar levels of 17β-estradiol, the reporter gene was up-regulated as judged by increased β-galactosidase activity. We also tested EDCs including bisphenol A and nonylphenol. And they could successfully be detected in the micromolar range using our system. All these together indicate that our bacterial cell-based biosensor can be used for the efficient and simple detection for the health-threatening EDCs.[This research was supported by the Civil research projects for solving social problems through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning (2015M3C-8A6A06012737)]

G047

Engineering Substrate Selectivity by Active-site Residues in Ferulic Acid Decarboxylase of Enterobacter sp. Px 6-4 by Site-saturation Mutagenesis

Sunil Ghatge, Youri Yang, and Hor-Gil Hur*

School of Environmental Science and Engineering, Gwangju Institute of Science and Technology

Conversion of lignin depolymerization products to value added chemicals has attracted increasing attention during last few years. Ferulic acid decarboxylase (FADase) catalyzes non-oxidative decarboxylation of lignin monomers p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, which is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various FADase, we selected the FADase from Enterobacter sp. Px 6-4, whose crystal structure is known, and tried to change the substrate selectivity using site saturation and site directed mutagenesis of active site residues Y27 and F95. Substitution of Y27S and F95E of FADase resulted into selective biotransformation of p-coumaric acid and ferulic acid in presence of mixture of the three substrates. The mutants characterized could be useful for the selective production of valuable chemicals such as 4-vinylphenol and 4-vinylguaiacol.


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H001

Antiviral Activity of Bisisomahanin from the Glycosmis stenocarpa

Jang Hoon Kim1, Ju Yeon Yoon1, Seung Kook Choi1, Sun Jung Kwon1, In Sook Cho1, Young Ho Kim2, and Gug Seoun Choi1*1Department of Horticultural Environment, National Institute of Horticultural and Herbal Science, RDA, 2College of Pharmacy, Chungnam National University

The growth and productivity of the peppers in field and greenhouse were suppressed by Pepper mild mottle virus (PMMoV). The aim of this study is to search for inhibitor on PMMoV from natural plants. Bisisomahanine was isolated from the aerial parts of Glycosmis stenocarpa (G. stenocarpa) by using Silica gel and C-18 column chromatography. It's structure was elucidated by 1H-NMR , 13C-NMR and Mass. This was revealed to have the inhibitory activity through the half-leaf assay against PMMoV. Furthermore, mixture of viral with compound was observed by electron microscope. This study suggested that bisisomahanine derived from G. stenocarpa may be the lead compound for the development of antiviral agents.[This study was supported by the basic research project (PJ010878012015) of Natural Institute of Horticultural and Herbal Science, RDA]

H002

Marine Fungal Resource Bank

Jae Young Park, Myung Soo Park, Ji Eun Eom, and Young Woon Lim*

Seoul National University

The Marine Fungal Resource Bank (MFRB), overseen by Dr. Young Woon Lim at Seoul National University, was designated as a marine bioresource bank of Korea by the Ministry of Oceans and Fisheries. The main goal of the MFRB is to establish a culture collection of marine fungi for educational, scientific, and industrial purposes. MFRB will undertake following tasks: 1) Survey marine environments across Korea to catalogue marine fungal diversity, 2) Establish a robust system of polyphasic species identification, 3) Evaluate the usefulness of the discovered fungi, 4) Create a secure preservation and loan system, 5) Provide web-based access to the database. With a global focus on utilizing natural resources, marine fungal resources provide excellent opportunities for educating the public on marine ecosystem, vitalizing marine research, and discovering novel substances for use as medicine and energy.[This work was supported by the Marine BioResource Bank Program of the Ministry of Ocean & Fisheries.]

H003

Pan-genomic Analysis of Lactobacillus salivarius Strains as Anti-pathogenic Swine Probiotics

Jun-Yeong Lee1, Geon Goo Han1, Gwi-Deuk Jin2, Sang Mok Lee1, Yun-Jaie Choi1, and Eun Bae Kim2,3*1Department of Agricultural Biotechnology, Seoul National University, 2Department of Animal Life Science, College of Animal Life Sciences, Kangwon National University, 3Division of Applied Animal Science, College of Animal Life Sciences, Kangwon National University

Probiotics is a promising candidate for replacement of antibiotics that were overused in the livestock industry, and in this aspect, antimicrobial activity against pathogens is the most important traits of probiotics. In the present study we attempted to identify genetic variety which affect its antimicrobial activity for selection of anti-pathogenic probiotics. We first isolated and identified the probiotic bacteria species, Lactobacillus salivarius that is used in broad field including human and livestock in the swine feces. Antimicrobial activity of isolated L. salivarius strains was measured against Escherichia coli and Salmonella enterica. L. salivarius strains that showed higher and lower bactericidal effect were selected and sequenced the entire genomes for pan-genomic analysis. We found some genetic variety between the lactic acid bacteria strains that showed higher and lower antimicrobial activity. These differences could be applied as genetic markers for selection of effective anti-pathogenic probiotics. [This work was supported by the Strategic Initiative for Microbiomes in Agriculture and Food, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea (914005-04). Yun-Jaie Choi and Eun Bae Kim are corresponding authors with equal contribution.]

H004

Development of Detection Method for Foodborne Pathogens at Low Density on Fresh Produce

Sujin Yun, Jin-Woo Park, Jae-Gee Ryu, and Sanghyun Han*

Microbial Safety Team, Department of Agri-Food Safety, National Institute of Agricultural Sciences

Foodborne illness outbreaks associated with fresh produce contaminated with human pathogens have resulted in an increasing demand for safe produce. Under even optimally controlled conditions, fresh produce could be contaminated with fecal matter entailing the potential presence of foodborne pathogens pre- and post-harvest. Population levels of the foodborne pathogens on fresh produce are thought to be very low because it is not the host for the pathogens. In this study, in order to develop a microarray that detects multiple pathogens present on fresh produce at very low level, three candidate sets of primers priming conserved regions within 16S rRNA shared by six different pathogens were tested for their ability to amplify low concentration of DNA samples. Amplified regions are variable in size and sequence for each pathogen. All of them were able to amplify their target region when DNA templates were diluted down to 0.001 ng/μl. Candiate #3 showed a better sensitivity compared to the others being able to amplify lower concentration of DNA. Bacterial cells were prepared at various concentrations, subjected to DNA isolation, and used in PCR reactions with the primer candidate #3. Cells at 5 × 101

CFU/ml were able to be detected when the PCR conditions were optimized. Next step will involve fresh produce inoculation with the tested pathogens. Probe design will be conducted targeting the variable region of the amplicon for each pathogen, which will help compose a microarray.

Poster

www.msk.or.kr | 73

H005

Relationship between the Microbiota in Different Sections of the Gastrointestinal Tract, and the Body Weight of Broiler

Geon Goo Han1, Jinyoung Lee2, Jun-Yeong Lee1, Gwideuk Jin3, Jongbin Park4, Changsu Kong2, Eun Bae Kim3, and Yun-Jaie Choi1*1Department of Agricultural Biotechnology, Seoul National University, 2Department of Animal Science and Technology, Konkuk University, 3Department of Animal Life Science, Kangwon National University, 4Department of Animal Life System, Kangwon National University

In the poultry industry, many efforts have been undertaken to improve the growth performance of broilers and identification and modulation of body weight (BW)-related bacteria could be one of the strategies to improve productivity. The aim of the study was to investigate the relationship between microbiota and BW in different sections of the gastrointestinal tract (GIT), and explore the weight related bacterial groups in broiler. A total of twenty 18-day-old birds were selected based on the BW, and samples were collected from the 3 different sections of the GIT, which included the crop, ileum and cecum. Bacterial genomic DNA was extracted from the samples, and the V4 region of 16S rRNA gene were amplified. Amplicons were sequenced on Illumina MiSeq, and microbial communities were analyzed by using QIIME. In principal coordinate analysis, bacterial communities were clustered into three groups, based on the sections of GIT. Several BW-related bacterial groups were identified from linear regression analysis. The results from the present study showed that particular bacterial communities in the GIT were related to BW, and the study has broadened the understanding of the intestinal microbial ecosystem in broiler. [This study was supported by the Strategic Initiative for Microbiomes in Agriculture and Food, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea (914005-04).]*Corresponding authors with equal contribution: Eun Bae Kim and Yun-Jaie Choi

H006

Prediction and Purification of Sesquiterpene Synthase from Wood Rot Fungus

Su-Yeon Lee, Jieun An, and MyungKil Kim*

National Institute of Forest Science

Terpenoid natural compounds have been exploited for drugs, fragrance and aroma ingredients. In our previous study, biosynthesis of eudesmane-type sesquiterpenoids which are composed with three isoprene unit, have been identified from wood rot fungus, Polyporus brumalis. In this study, transcriptome analysis has been conducted to understanding the terpenoid metabolism in P. brumalis. RNA sequencing of P. brumalis was performed using PacBio next generation sequencing suing Single Molecule Real Time. Non-redundant transcripts (11,143) obtained by isoforms statistics analysis were annotated as BLASTX result against GO database. Three sesquiterpene synthases has been predicted from three wood rot fungi, Gloephyllum trabeum, Dichomitus squalenes and Trametes versicolor. Differentially expressed genes (DEGs) analysis of annotated genes has been performed in the three mycelium which were inoculated in cultures of control, magnesium blank and phosphate blank. As the results, the highest expression level of G. trabeum sesquiterpene synthase was shown in phosphate blank culture. Also, the yield of eudesmane type sesquiterpenoids was obtained from phosphate blank culture. To investigate a gene function of predicted sesquiterpene synthases on terpene metabolism of P. brumalis, purification and characterization of sesquiterpene synthase have being researched.

H007

Genetic Analyses of Sinonovacula constricta from Southern Coast of Korea Based on DNA Sequences of Mitochondrial Cytochrome Oxidase I Gene

Mi Sun Kim, Ji Hee Lee, Joo Won Kang, Dae In Kim, and Chi Nam Seong*


Sinonovacula constricta is a benthic clam and one of the important economic shellfish. The mitochondrial cytochrome oxidase subunit 1 (CO I) has been widely used in genetic diversity and population genetic structure of marine species. In this study, we compared the partial sequences of the CO I gene of Sinonovacula constricta. Samples were collected from Goheung and Beolgyo on the southern coast of Korea. Ten haplotypes were identified out of 30 individuals. Four haplotypes (A, B, C, D) were recovered from the both regions. The most frequent haplotype B consisted of 12 individuals, including seven from Goheung and five from Beolgyo. 4 haplotypes (E, F, G, H) were found only in Beolgyo, but 2 haplotypes (I, J) also occurred in Goheung.[This work (Grants No. C0268343) was supported by Business for Academic-industrial Cooperative establishments funded Korea Small and Medium Business Administration in 2015.]

H008

Evaluation of Bacterial Community Composition of Commercial Animal Probiotics from Korea Using Brcoded Pyrosequencing

Baolei Jia, Hyo Jung Lee, and Che Ok Jeon*


A wide variety of probiotics for animal feeding have been used because the use of antibiotics for animal as growth promoters was prohibited. However, just few studies have been conducted to evaluate the microbial compositions for commercial probiotic products, whether labeling on the products for animal feeding represents the true microbiological contents or not. Bacterial community compositions of fifty commercial probiotics of twenty-six brands in Korea were investigated using a barcoded pyrosequencing approach. Pyrosequencing data were processed and classified using RDPipeline and the presence of potential pathogenic bacteria were estimated by Blastn based on PHI (Pathogen Host Interaction) database. Labeling on the products showed that Lactobacillus, Bacillus, Clostridium, and Saccharomyces were major components. The pyrosequencing data indicated that some probiotics were relatively well accordance between bacterial claims on their labels and classification results. However, some differences were also observed in some probiotic products; some probiotic products even contained not declared bacteria. About 2% of the total sequences were closely related to potentially pathogenic sequences such as Pseudomonas aeruginosa, Burkholderia cenocepacia, and Escherichia coli. [This study was supported by the ‘Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01090604)’ of Rural Development Administration, Republic of Korea.]


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H009

A Phylogenic, Functional and Metabolic Analysis of Bacillus velezensis Using Pan Genome

Byung Hee Chun and Che Ok Jeon*


Bacillus velezensis is a rod-shaped, spore-forming and Gram-positive bacterium that grows under strictly aerobic conditions. In this study we performed the pan and core genome analysis of B. velezensis to investigate phylogenic, functional and metabolic features of B. velezensis. We retrieved all genomes (46) of B. velezensis and its relatives (named as B. velezensis, B. amyloliquefaciens, B. methylotrophicus, and B. siamensis in GenBank) from GenBank and checked their qualities using CheckM. Average nucleotide identity analysis of eventually obtained 46 genomes showed that 40, 4, and 2 genomes were classified into B. velezensis, B. amyloliquefaciens, and B. siamensis, respectively and a phylogeny tree using 2054 core genes also supported that they were clearly split into different phylogenetic lineages–– a phylogenetic analysis based on 16S rRNA gene sequences did not clearly differentiate them. The genome analysis of forty B. velezensis strains was found to contain 5932 pan genes and 2490 core genes. COG distribution analysis using core genome revealed that B. velezensis contains high fractions for amino acid transport and metabolism and transport categories and B. velezensis harbors various antimicrobial activity and salt tolerance-related genes.

H010

Pan-genome Analysis of Leuconostoc mesenterides Reveals Its Metabolic Capabilities and Diversities during Fermentation

Hye Hee Jeon and Che Ok Jeon*


To investigate the comprehensive characteristics of Leu. mesenteroides, we performed the pan-genome analysis using whole genome sequences of sixteen Leuconostoc (Leu.) mesenteroides strains available in GenBank database–– average nucleotide identity values also supported that all they belong to Leu. mesenteroides, The pan-genome of the sixteen Leu. mesenteroides strains was found to contain 3088 genes and 716 core genes. A phylogenetic tree was constructed using all core genes to investigate their phylogenetic relationships, which were a little different from the phylogenetic relationships based on 16S rRNA gene or house-keeping gene sequences and they could be grouped into four subspecies. For the analysis of functional capabilities of Leu. mesenteroides, all protein sequences were assigned into respective COG categories. The COG analysis showed that Leu. mesenteroides species have high fraction for transport and metabolism of carbohydrates and amino acids, which is similar with other Leuconostoc species. In addition, to investigate metabolic capability of Leu. mesenteroides, all functional genes of sixteen genomes were mapped onto the KEGG pathways. As the results, genes associated with carbohydrate and amino acid metabolism and PTS systems related to sugar transports were abundant, suggesting that Leu. mesenteroides was evolved to adapt to environments with various carbon sources. This is the first study to investigate the metabolic properties of Leu. mesenteroides.

H011

Antimicrobial Activity of Essential Oil of Eucalyptus globulus against Fish Pathogenic Bacteria

Joon-Woo Park, Mitchell Wendt, Sabrina Hossain, Sudu Hakuruge Madusha Pramud Wimalasena, and Gang-Joon Heo*

College of Veterinary Medicine, Chungbuk National University

The antibacterial activities of the essential oil of Eucalyptus globulus (EOEG) was determined against 7 fish pathogenic bacteria (Edwardsiella tarda, Streptococcus iniae, S. parauberis, Lactococcus garviae, Vibrio harveyi, V. ichthyoenteri and Photobacterium damselae) obtained from farmed olive flounder. The inhibitory activity was evaluated by three methods: Disc diffusion method, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). According to the disc diffusion test, as the concentration of EOEG (5-40 μg) rises, the inhibitory zone increases in size. Compared with amoxicillin, tetracycline and chloramphenicol, EOEG showed similar antibacterial activity. The MIC of EOEG ranged from 7.8 to 125 mg/ml and MBC values ranged from 62 to 250 mg/ml. These results show that EOEG has antimicrobial activity against all seven bacteria, but there was no marked difference between each genus. From these results, it is suggested that EOEG can be used as an antimicrobial agent against fish bacterial diseases in the fish industry.

H012

Morphological Changes of MG-63 Cells by Fucoidan Treatment

Hyeseon Kim and Taeck J. Jeon*


Fucoidan, a fucose-rich sulfated polysaccharide which is found in brown algae, possesses diverse biological activities including antibacterial, antioxidant, anticoagulant, anti-inflammatory, and antitumor activities. Recently, the fucoidan has been reported to induce apoptosis in various cell lines. In this study, the apoptotic effects of fucoidan were investigated by analyzing morphological changes in MG-63 osteosarcoma cells in the presence of fucoidan. We founded that the viability and proliferation of MG-63 cells was decreased in the presence of fucoidan in a dose- and time-dependent manner. Fucoidan-treated cells became shrunk and rounded. The cells were highly aggregated and much smaller than control cells. To further understand these morphological changes of the cells in the presence of fucoidan, we examined the fragmentation of DNA and apoptotic effects of fucoidan. When the cells were treated with fucoidan, some fragmented DNAs were observed while there was no such fragmented DNA in the absence of fucoidan. In addition, significantly increased number of cells was stained by AnnexinV in the presence of fucoidan, compared to control cells. Our results suggest that fucoidan has apoptotic effects on MG-63 cells with morphological changes.[This research was surpported by Basic Science Research Program through NRF.]

Poster

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H013

Effectiveness of Periodic Treatment of Quercetin against Viral Hemorrhagic Septicemia Virus

Se-Young Cho1, Yeong O Kim2, Se Hyun Cho2, Jong-Soon Choi3, Joseph Kwon3, and Duwoon Kim1,2*1Foodborne Virus Research Center, Chonnam National University2Department of Food Science and Technology, Chonnam National University3Korea Basic Science Institute

Viral hemorrhagic septicemia virus (VHSV), a rhabdoviral fish pathogen, creates a major threat to the development of aquaculture industry worldwide. The fathead minnow (FHM) cells infected with VHSV at a multiplicity of infection (MOI) of 0.2 and treated with 100 μl quercetin. The quercetin treatment showed cell viability of 64.3% at 42 h post infection, while non-treated cells infected with VHSV at the same MOI had a cell viability of 23.7%. The highest cell viability was found by the treatment of quercetin (Q) and followed by kaempferol (K), taxifolin (T), catechin (C), myricetin (M), quercetin 3-O-α-L-arabinoside (Q3A), quercetin 3-O-rutinoside (Q3R), quercetin 3-β-D-glucuronide (Q3Glu), quercetin 3-β-glucoside (Q3G), kaempferol 3-O-β-D-glucoside (K3G) and quercetin 3-β-D-galactoside (Q3Gal). In this study, the effect of pretreatment and periodic treatment with quercetin against VHSV in FHM cells. Compared to pretreatment, periodic treatment resulted in significantly higher cell viability but lower relative expression of the VHSV titration and total apoptosis and cell death. In conclusion, the scope of quercetin application could be open up as a new prospective for the development of the therapeutic treatment of viral hemorrhagic septicemia. [Supported by grants from the Center for Analytical Research of Disaster Science of Korea Basic Science Institute.]

H014

A Functional Annotation System Based on Functionally Equivalent Protein/Domain Search Algorithm

Dong Su Yu

National Institute of Ecology

In a functional annotation system, homology-based transfer (HBT) is a gold standard for transferring the functional description of homologs to the putative proteins. To search homologs for the putative proteins, sequence-based methods such as BLAST and HMMER have been commonly used because of fast homolog searches against big databases. Although many putative proteins have been annotated by HBT using sequence-based methods with high confidence, meticulous attention is still required owing to the emergence of misannotated or unannotated proteins caused by missing functionally equivalent homologs. Actually, the performance of HBT-based annotation system using sequence-based methods is considerably dependent upon the ability of detecting functionally equivalent homologs. We developed an HBT program based on functionally equivalent proteins (FEPs) or domains (FEDs) using the FEP-BH algorithm, called HBT-FEPD. As FEP-BH algorithm is used to detect FEPs from the outputs of blastp and hmmsearch, HBT-FEPD can more precisely annotate the putative proteins by the selected FEPs and FEDs.

H015

Anti-Norovirus Activity from Natural Plant Extract Using a Norovirus Surrogate System

Joo Bong Choi1, Diana Soils Sanchez1, Hee Jung Lee2, In Sun Joo2, Jeong Su Lee2, and Sung-Joon Lee1*1Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University

2Food Microbiology Division, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation

Norovirus (NoV) has been identified as a major cause of acute gastroenteritis epidemics worldwide. This study investigated the effect of Extract A on the infectivity and viral replication of norovirus. Murine norovirus (MNV), a surrogate of human norovirus, was pre-incubated at titers of ~5 log10 PFU/ml with Extract A and then used to infect RAW 264.7 cells in a plaque reduction assay. MNV incubated with Extract A at 4°C exhibited a significant reduction in MNV plaque formation in both time- and dose-dependent manners. qPCR results revealed that the viral RNA in the RAW 264.7 host cells infected with Extract A-treated NMV was significantly reduced compared to that in cells infected with untreated MNV. These results suggest that Extract A inactivates norovirus and its subsequent replication in host cells. Thus, Extract A shows promise as method of inhibiting norovirus within the food industry.[Supported by grants from KFDA]

H016

The Role of Sir2 in Oxidative Stress Response Changes Depending on the cAMP-PKA Signaling

Yeong Hyeock Kim, Woo Sun Song, Woo Kyu Kang, and Jeong Yoon Kim*

Department of Microbiology and Molecular Biology, College of Bioscience and Biotechnology, Chungnam National University

Silent Information Regulator 2 (Sir2), which is a NAD+-dependent protein deacetylase, is known to contribute to calorie restriction (CR)-mediated lifespan extension in yeast. However, the link between Sir2 and oxidative stress in CR-mediated lifespan extension remains elusive. We have recently reported that the role of Sir2 in oxidative stress resistance and chronological lifespan is dependent on growth phase in yeast. In this study, we show that the cAMP-PKA signaling determines the Sir2’s role in oxidative stress response. Deletion of PDE2, which increases cAMP level, changed the negative role of Sir2 for H2O2 resistance to the positive during early exponential phase. In contrast, deletion of RAS2, which reduces cAMP level, reversed the positive role of Sir2 for H2O2 resistance to the negative during post diauxic phase. Additionally, buffering of culture medium to pH 6.0, which is known to inactivate the Ras/cAMP-PKA pathway, made sir2 mutant cells more resistant to H2O2 than wild type during post-diauxic phase. We next examined whether expression of CTT1 encoding cytoplasmic catalase is regulated by Sir2 in a cAMP-PKA signaling-dependent manner. The changes in the CTT1 expression level by PDE2 or RAS2 deletion were in agreement with those of the H2O2 resistant phenotypes. Collectively, this study suggests that Sir2 is involved in the regulation of genes for oxidative stress resistance, negatively or positively depending on the activity of the cAMP-PKA signaling pathway.


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H017

Immunogenicity and Efficacy of VLP Forming Baculoviral Vaccine against Influenza pdmH1N1 in BALB/c Mice

Yong-Dae Gwon, Sehyun Kim, Yoonki Heo, Hansam Cho, Yeondong Cho, Ki Hoon Park, Yuyeon Jang, Jong Kwang Yoon, Hee-Jung Lee, and Young Bong Kim*

Department of Bio-industrial Technologies, Konkuk University

An outbreak of influenza H1N1 in 2009, representing the first influenza pandemic of the 21st century, was transmitted to over a million individuals and claimed 18,449 lives. The current status in many countries is to prepare influenza vaccine using cell-based or egg-based killed vaccine. However, traditional influenza vaccine platforms have several limitations. To overcome these limitations, many researchers have tried various approaches to develop alternative production platforms. One of the alternative approach, we constructed a human endogenous retrovirus (HERV) envelope-coated, baculovirus-based, virus-like-particle (VLP)–forming DNA vaccine (termed AcHERV-VLP) against pandemic influenza A/California/04/2009 (pH1N1). BALB/c mice immunized with AcHERV-VLP (1×107 FFU AcHERV-VLP, i.m.) and compared with mice immunized with the killed vaccine or mice immunized with AcHERV-HA. As a result, AcHERV-VLP immunization produced a greater humoral immune response and exhibited neutralizing activity with an intrasubgroup H1 strain (PR8), elicited neutralizing antibody production, a high level of interferon-γ secretion in splenocytes, and diminished virus shedding in the lung after challenge with a lethal dose of influenza virus. In conclusion, VLP-forming baculovirus DNA vaccine could be a potential vaccine candidate capable of efficiently delivering DNA to the vaccinee and VLP forming DNA eliciting stronger immunogenicity than killed vaccines.[Supported by grants from iPET]

H018

Enhanced Immune Response for Foot-and-Mouth Disease Virus Vaccine with Granulocyte Macrophage Colony Stimulating Factor-flagellin Adjuvant

Yu Yeon Jang Jang1, Hansam Cho1, Yong-Dae Gwon1, Hanul Choi1, Jaehyuck Heo1, Gwonsung Joo1, Joong-Bok Lee2, Jiwon Choi1, and Young Bong Kim1*1Department of Bio-industrial Technologies, Konkuk University, 2Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University

Foot-and-mouth disease (FMD) is a highly infectious and economically devastating disease of cloven-hoofed animals. Here, this study was utilized Granulocyte-macrophage colony-stimulating factor (GmCSF) and Salmonella typhimurium Flagellin 2 (STF2) as adjuvant. GmCSF-STF2 fusion was constructed a recombinant baculovirus, which is connected 2A-linker peptides and encoding adjuvant (Ac-ie1-PERV-GmCSF-STF2). GmCSF and STF2 genes were confirmed gene expression levels by reverse transcription PCR in PK15 cells. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), which is induced by Toll-like-receptor 5 (TLR5) signaling pathway, was detected by reverse transcription PCR in PAM cells. To investigate the effects as adjuvant, immunized with commercial FMD vaccine in mice. The immunological effects of the Ac-ie1-PERV-GmCSF- STF2 were determined specific FMDV by ELISA, ELISPOT, and CTL assay. GmCSF-STF2 fusion showed higher IgG titer, INF-γ secretion, and CTL assay than that of FMD vaccine only. Extending these result, GmCSF-STF2 could stimulate both humoral and cell-mediated immune response as an adjuvant for FMD vaccine. In conclusion, STF2 and GmCSF could be useful for potential vaccine adjuvant against pathogen.[This research was supported by Fishery by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries) from Ministry of Agriculture.]

H019

Chronic Repression of mTOR Complex 2 Alters the Composition of the Gut Microbiota in Diet-induced Obese Mice

Mi-Ja Jung, Jina Lee, Min-Soo Kim, Dong-Wook Hyun, Na-Ri Shin, Ji-Hyun Yun, Pil Soo Kim, Tae Woong Whon, and Jin-Woo Bae*


Alterations in the gut microbiota play a crucial role in host physiology and metabolism; however, the molecular pathways underlying these changes in diet-induced obesity are unclear. Mechanistic target of rapamycin (mTOR) signaling pathway is associated with metabolic disorders such as obesity and type 2 diabetes (T2D). Therefore, we examined whether changes in the regulation of mTOR signaling induced by diet (a high-fat diet [HFD] or normal-chow diet) and/or therapeutics (resveratrol [a specific inhibitor of mTOR complex 1] or rapamycin [an inhibitor of both mTOR complex 1 and 2]) altered the composition of the gut microbiota in mice. Oral administration of resveratrol prevented glucose intolerance and fat accumulation in HFD-fed mice, whereas rapamycin significantly impaired glucose tolerance and beta cell function. The abundance of Lactococcus, Clostridium XI and Hydrogenoanaerobacterium increased under HFD condition; however, the abundance of these species declined after resveratrol treatment. Conversely, the abundance of unclassified Marinilabiliaceae and Turicibacter decreased in response to a HFD or rapamycin. Taken together, these results demonstrated that changes in the composition of intestinal microbiota, resulted from differential mTOR activity, correlated with obese and diabetic phenotypes, suggesting mTOR pathway as a novel therapeutic target for obesity and T2D. [Supported by grants from NRF and MAFRA.]

H020

Effects of Freeze-drying Feces on 16S rRNA Based Microbial Community Analysis

Jungman Kim, Nakwon Hwang, Mincheal Kim, Son G. Nguyen, and Tatsuya Unno*

Faculty of Biotechnology, College of Applied Life Science, SARI, Jeju National University

Since next generation sequencing (NGS) was developed, microbial communities in intestine and environments have been widely investigated. Especially, gut microbiota studies have been published number of times. These studies have suggested that gut microbiota was related to intestinal diseases such as obesity, diabetes, diarrhea and cancer. While these results were obtained from DNA sequences obtained from fecal samples, previous studies have reported that results could be varied based on DNA extraction methods, DNA concentrations used in PCR, PCR conditions, and sample homogenization. Freezing drying fecal samples prior to DNA extraction is an ideal method for effective DNA extraction, consistent fecal amount, sample homogeneity, and long term storage. In this study, we examined if freeze-drying fecal samples affect sequence-based microbial community analysis by comparing microbial communities of fresh and freeze-dried fecal samples. Our results showed that number of rare operational taxonomic units (OTUs) was 1.2–1.5 times lower in freeze-dried feces than that of fresh feces while no significant differences were observed between overall community analysis. Substantial rare OTUs are known artificial errors in MiSeq, thus reduced number of rare OTUs indicate better sequencing quality, suggesting that freeze-drying feces may not only provide benefits such as long-term storage and better homogenization, but also increases quality of MiSeq-based microbiota analysis results.

Poster

www.msk.or.kr | 77

H021

The Roles of Histone H3-K4 Methylation in Morphogenesis of Candida albicans

Jueun Kim and Jung-Shin Lee*

Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University

Candida albicans is the most common fungal pathogen in human and its ability to grow as a filamentous form is important for its virulence. In hyphal formation of C. albicans, the transcriptional activation of hypha-specific genes is very important. The methylation of histone H3 at lysine 4 (H3K4) is an epigenetic mark studied extensively because it is related to transcription by RNA polⅡ. Here, we observed that the Δset1 strain, a deletion mutant of H3K4 methyltransferase gene, develops hyphae more rapidly. To identify the relationship between H3K4 methylation and the transcription of hypha-specific genes in C. albicans, we performed the RNA-seq of Δset1 strain in normal condition or hyphal induced condition. The expression levels of hypha-specific genes in Δset1 strain were more increased than those of WT. To investigate the precise role of H3K4 methylation in C. albicans morphogenesis, we analyzed the localization of each H3K4 methylation status in each condition. Surprisingly, we found that the H3K4me2 and me3, generally localized at active transcribed genes, is not detected in most of hypha-specific genes in any conditions. However, the level of H3K4me1, whose role is not much known, is enriched at hypha-specific genes in normal condition and increased in hyphal formation. These results show that the H3K4 methylation regulates hyphal formation of C. albicans by unconventional regulation mechanism. [This work is supported by NRF-2014H1A2A1021300 and NRF-2015R1A-4A1041105]

H022

Histone Residues Play Important Roles for HM Silencing Maintenance in Saccharomyces cerevisiae

Soojin Yeom and Jung-Shin Lee*

Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University

Gene silencing is one of important concepts for the epigenetic gene regulation. Histone modifications are critical factors for the maintenance of gene silencing in eukaryotic systems. However, none of histone modifications as epigenetic silencing markers is conserved in Saccharomyces cerevisiae. To identify novel global epigenetic silencing marker from yeast to human, we developed screening method using the system for the maintenance of yeast mating type. We used yeast histone library, which is a collection of strains containing alanine-substituted histone residue, and yeast single-gene knockout library. From this screeing, we found that the 79th lysine of histone H3 (H3K79) is required for the maintenance HM silencing, but Dot1, methyltransferase of hisotne H3K79 is not. Also we checked several histone residues are required for the maintenance of HM silencing in S. cerevisiae. [This work is supported by NRF-2015R1D1A1A02061743 and NRF-2015-R1A4A1041105]

H023

PAF1 Complex Directly Regulates H3K4 Methylation in Saccharomyces cerevisiae

Jun-Soo Oh and Jung-Shin Lee*

Molecualr Biochemistry Lab, Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University

In eukaryotes, many aminoacid residues of histone components can be easily modified by many histone modifying enzymes and this histone modifications are very important in transcriptional regulation. H3 Lys4 methylation can be conducted in mono, di, and tri. Monoubiquitination in H2B at K120(K123 in yeast) is prerequisite for di- and tri-methylation of H3K4. In other words, histone crosstalk between this two modifications exist. It is reported that PAF1 complex contributes to the H2B monoubiquitination. In yeast, PAF1 complex consists of PAF1, LEO1, CDC73, CTR9 and RTF1. This complex is structurally, functionally well conserved from yeast to human. It is already reported that PAF1 complex is important in regulating H2B monoubiquitination and indirectly regulates the H3K4 di- and tri-me-thylation by histone crosstalk. But, we suggests that PAF1 complex directly regulates the H3K4 methylation, in addition to the reported indirect manner.[Supported by grants from NRF-2015R1D1A1A02061743 and NRF-2015-R1A4A1041105]

H024

Korea National Microorganisms Research Resource Center

Se Joung Yeom and Sang Seob Lee*

Kyonggi University

The Korea National Microbiological Research Resource Center is the core center of the twelve microorganism banks designated by the Ministry of Education, Science and Technology. The KNMRRC supports microorganism banks with necessary guidelines, standards, training for efficient operation of the banks. It also provides with an effective forum to solve common issues of the related banks. The ultimate goal of the KNMRRC is the followings: 1-construction of standardized and integrated management system, 2-construction of Core center and other organs network, 3-Quality Control (QC) of microbial resources in the member banks, 4-conservation of Resources in the member banks and the interrupted banks, 5-education for professionals in the member banks, 6 public Relations for raising people's awareness of the importance of microbiological resources.


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H025

Center for Fungal Genetic Resources (CFGR): Housing Plant Pathogenic Fungi for Educational and Research Purposes

Yeo Kyoung Yoon and Yong-Hwan Lee*

Center for Fungal Genetic Resources, Seoul National University

Fungi are eukaryotic organisms, growing in a wide range of habitats. Fungi are significantly important in a variety of ways. They play an essential role in the decomposition of organic matter. They have been used as a source of food, and agents for fermentation of food products and for the production of various antibiotics and enzymes that are used in a field of research, industry, medicine, etc. In contrary, impact of many fungi on animals and plants is economically and socially detrimental. For example, Magnaporthe oryzae causes the most destructive disease, “rice blast”. Annual yield loss of rice by rice blast is equivalent to rice that could feed about 60 million people. The Center for Fungal Genetic Resources (CFGR) was established to collect, maintain and distribute genetic resources mainly from plant pathogenic fungi, which are important for both educational and research purposes. This will contribute to development of new strategies for management of crop diseases and of new components for improvement of our lives. CFGR possesses important fungal species; a total of 42,000 isolates from 54 species of fungi including 20,902 T-DNA transformants of rice blast fungus and anthracnose fungus. In addition to the biological materials, CFGR has developed user-friendly databases to maintain genetic information of fungal stocks and help to solve questions about fungal pathogenicity, population genetics, development, and evolution. Also, CFGR seeks strategies for sustainable and scientific plant quarantine to better protect our ecosystem from invasive microorganisms.

Keywords: Plant pathogen, Fungi, Mutant, Plant quarantine, Pathogenicity genes, Genetic resource

H026

Korean Metagenome Bank for Exploiting Microbial Diversity

Jung-Hoon Yoon

Department of Food Science and Biotechnology, Sungkyunkwan University

Microorganisms have played important roles in biotechnology and bioindustry for long times. The recent use of molecular ecological methods and environmental DNA (eDNA) has changed our knowledge of microbial diversity dramatically and provided rapid access to genes of yet-uncultured microorganisms. Application of molecular ecological studies has shown that the majority (99%) of microorganisms present in the nature are under uncultivation. Many attempts to improve the recovery of microorganisms and their genes from the environmental samples have recently been achieved. Metagenomic approach that recovers the environmental DNA without the limitations of culture-dependent methods and constructs DNA libraries in suitable cloning vectors and host strains have been utilized for retrieving novel and useful genes. Korean Metagenome Bank (KMGB), a member of Korea National Research Resource center (KNRRC), has opened with the goal for the collection and distribution of metagenome (eDNA) and metagenomic library. The aims of the Korean Metagenome Bank are to contribute to the development of biotechnology by providing the metagenomic resources into various researches and to perform a national mission for maintaining the metagenomes as future biological resources

Keywords: Metagenome, library, environmental DNA

H027

Microbial Carbohydrate Resource Bank (MCRB)

Seunho Jung

Department of Bioscience and Biotechnology, Microbial Carbohydrate Resource Bank(MCRB) & Center for Biotechnology Research in UBITA (CBRU), Konkuk University

Microorganisms have played important roles in biotechnology and bioindustry for long times. The recent use of molecular ecological methods and environmental DNA (eDNA) has changed our knowledge of microbial diversity dramatically and provided rapid access to genes of yet-uncultured microorganisms. Application of molecular ecological studies has shown that the majority (99%) of microorganisms present in the nature are under uncultivation. Many attempts to improve the recovery of microorganisms and their genes from the environmental samples have recently been achieved. Metagenomic approach that recovers the environmental DNA without the limitations of culture-dependent methods and constructs DNA libraries in suitable cloning vectors and host strains have been utilized for retrieving novel and useful genes. Korean Metagenome Bank (KMGB), a member of Korea National Research Resource center (KNRRC), has opened with the goal for the collection and distribution of metagenome (eDNA) and metagenomic library. The aims of the Korean Metagenome Bank are to contribute to the development of biotechnology by providing the metagenomic resources into various researches and to perform a national mission for maintaining the metagenomes as future biological resources

Keywords: Metagenome, library, environmental DNA

H028

Bacteriophagebank

KyoungEun Cha and Heejoon Myung*

Dept. of Bioscience and Biotechnology, Hankuk University of Foreign Studies

Bacteriophages are viruses growing on bacterial hosts. They are antagonistic to bacteria and first reported by Frederick Twort and Felix d'Herelle in 1915 and 1917, respectively. They are found in sea, air, land and even foods. It is assumed that 1030 to 1032 phages exist on earth and they play a role in maintenance of biological balance. Recently, new applications for phages are increasingly reported. As they are a part of useful biological resources, there are increasing demands for securing these resources. In response to these demands, the bacteriophage bank was established in 2010. The bank collects phages from environments as well as from working groups worldwide. Currently, 600 different phages are stocked. The host bacteria include E. coli, Salmonella enteritidis, Pseudomonas aeruginosa, Listeria monocytogenes, Acinetobacter, Camphylobacter jejuni, Enterococcus faecium, Enterococcus faecalis, Cronobacter sakazakii, Seratia marcescens and Staphylococcus aureus. The number of stock is growing continuously. The bank also serves as a distributor for the collected phages. (www. phagebank.or.kr)

Keywords: Bacteriophage, 600 different phages, useful biological resources

Poster

www.msk.or.kr | 79

H029

Korea Mushroom Resource Bank

Jae Young Park, Nam Kyu Kim, Hey Young Choi, Mi Jin So, and Young Woon Lim*

Seoul National University

The Korea Mushroom Resource Bank (KMRB) was launched as a national research resource bank in 2015 by the Ministry of Science, ICT and Future Planning. The main goal of the KMRB is to secure important biological resources, mushroom-forming basidiomycota, significant sources of fun-damental and novel substances and materials, as dried specimen, cultures, and genomic DNA. For wider application of fungal resources in education, medicinal and industrial uses, the KMRB will undertake following tasks: 1) Survey natural environments across Korea to catalogue mushroom diversity, 2) Establish resource management system based on accurate identification of mushroom, 3) Evaluate the usefulness of the discovered mushroom, 4) Create a secure preservation and loan system. With a global focus on utilizing natural resources, mushroom resources provide excellent opportunities for academic research, and discovering novel substances for use as medicine and energy.

Keywords: Korea Mushroom Resource Bank, Mushroom, Fungi, Culture, Collection

H030

Korea Bank for Pathogenic Viruses

Ki-Joon Song

Korea Bank for Pathogenic Viruses

Korea Bank for Pathogenic Viruses (KBPV) has been established in 2005 as a repository agent for the collection, management and distribution of the various pathogenic viruses that are essential to use for researches in biomedical sciences. The Institution operates in collaboration with The Institute for Viral Disease at Department of Microbiology, College of Medicine, Korea University, founded in 1973.The bank has unique viral collections such as Hantaan, Seoul, Muju, Soochong, and imjin the etiologic agents of hemorrhagic fever with renal syndrome. To date, total of more than 43,000 materials (~100,000vials) from human and animal have been collected and maintained.We have provided a highly collaborative environment for researchers in various fields by providing valuable viral resources including consulting service. We also provide the educational program related to pathogenic viruses including biosafety training. Requestors of such agents are required to register with KBPV and to supply details of their laboratory facilities and safety management. More details about KBPV can be found at ; http://kbpv.knrrc.or.kr

Keywords: pathogenic viruses, biosafety, viral disease, genetic information, antibody, biomaterial, vaccine, epidemiology, public health

H031

Plant Virus GenBankKi Hyun Ryu

Dept. of Horticulture, Biotechnology and Landscape Architecture

Plant Virus GenBank (PVGB) is a nonprofit semi-governmental organization, one of the Korea National Research Resources Collections (KNRRC) for special research materials Banks program financially supported by the Ministry of Education, Science & Technology (MEST) dedicated to collection, identification, characterization, preservation, research development, distribution and deposition of plant virus research biomaterials established since 1999. PVGB is one of substructure of Korea National Microbiological Research Resources Collections (KNMRRC). PVGB retains a number of accessions and a wide range of collections of Plant Virus Biomaterials useful for Plant Virology and Biotech-related research areas. PVGB has moved to its current status on November in 2000 and has modern facilities and infrastructures for supporting broad research fields as well as Plant Virology Community. PVGB has been recognized as a member of World Federation for Culture Collection & World Data Center for Microorganisms (WFCC-WDCM) and ISBER since April of 2001 and June of 2007, respectively. Main objectives and contents of PVGB can be categorized as 7 topics as follow ; collection and development of Plant Virus Research Biomaterials such as infectious plant virus culture, plant viral cDNA clone, plant virus antiserum, biologically active full-length cDNA clone, viral cDNA library, virus-induced plant cDNA library, and diagnostic primers, preservation of Plant Virus Research Biomaterials, Distribution of Plant Virus Research Biomaterials to worldwide researchers to support their research fields and Safe Deposit from virologists, Development of New Plant Virology Techniques, i.e., molecular taxonomy of plant viruses, infectious cDNA clones, molecular indexing of virus variation, screening of virus resistance, virus-resistant transgenic plants, and risk assessment for living modified (LM) virus and LM plant systems, collection and support of Research Information.

H032

Lichen as a Novel Bioresources in Korea

Young Jin Koh and Jae-Seoun Hur*

Korean Lichen Research Institute, Sunchon National University

Lichens are symbiotic organisms composed of a fungus (mycobiont) and an alga (photobiont). They produce characteristic secondary metabolites, lichen substances, which seldom occur in other organisms. Lichen and their metabolites have many biological activities. In spite of the wide spectrum of biological activities shown by the lichens, they have long been neglected by mycologists and overlooked by agrochemical industry because of its slow growth in nature and difficulties in the artificial cultivation of organisms. Use of lichen-forming fungi can overcome the disadvantage of natural lichen extracts for industrialization of their metabolites because of their much faster growth and larger production of the metabolites in culture than the natural thalli. Korean Lichen and Allied Bioresources Center focuses on isolation, maintenance and distribution of lichen bioresources to research groups in universities, national institutes and industrial sectors. It also screens their biological activities, and investigates cultural conditions for large production of lichen substances. Chemical library of some lichen extracts is also available from the center.

Keywords: lichen, lichen-forming fungi, photobiont


80 | www.msk.or.kr

H033

Korean Collection for Oral Microbiology

Soon-Nang Park, Yun Kong Lim, Eojin Jo, and Joong-Ki Kook*

Department of Oral Biochemisty, School of Dentistry, Chosun University

It has been known that about 700 species of oral bacteria inhabit the human oral cavity. Of them, 350 species have been cultured. The oral bacteria are the major causative agents of systemic diseases such as cardiovascular diseases as well as oral diseases, periodontitis and dental caries. However, the causative bacterial species for oral diseases have not been known because the dental diseases are occurred by the multiple infections. In addition, the prevalence of the oral bacterial species is different by the geographic location of the host and individual. It is very important to obtain the oral bacteria from Koreans for pathogenesis studies related to oral infectious diseases. The purpose of Korean Collection for Oral Microbiology is to obtain the oral clinical strains and their genetic resources, such as 16S rDNA, species-specific PCR or qRT-PCR primers, and genome sequences, for offering them to the researchers.

H034

Culture Collection of Antimicrobial Resistant Microbes

Hyunjin Hong, Hakmi Lee, Minyoung Lee, Yeonhee Lee, and Eunju Shin*

Culture Collection of Antimicrobial Resistant Microbes, Department of Biology, Seoul Women’s University

Today, the increasing clinical abuse of antimicrobials in people and animals, led to a high rate of occurrence of resistant microbes. In addition, drug resistance is easily transferred from one resistant species to another related one in many ways, thereby complicating the issue. Therefore, treatment for disease caused by antimicrobial resistant microbes has emerged as a critical issue worldwide, and development of new drugs that inhibit resistant microbes became an urgent issue of research. As the issue should be dealt across clinical research, regulation, and pharmaceutical development, communication and cooperation between researchers among these areas are necessary.Since Culture Collection of Antimicrobial Resistant Microbes was established in 1999, CCARM has been played a role as a connector among various research fields by providing the antimicrobial resistant microbes with known mechanism and information. CCARM collects, keeps, and preserves the resistant microbes in a systemic manner for constant supply of certified microbes and share the information with researchers in various fields. CCARM has a collection of over 20,000 strains of bacteria and yeast from 87 genera and provides various information including international meeting, newest information related to resistance via homepage and newsletter. CCARM is now increasing the interaction and collaboration between culture collections through national and international network as a member of Clinical Laboratory Standards Institute since 2000, World Federation for Culture Collection & World Data Center for Microorganisms since 2003, International Society of Biological and Environmental Repositories since 2007, Korea National Research Resource Center since 2008, and Biological Repositories since 2009.

Keywords: antimicrobial resistant, research resource center, bacteria

H035

Korea Environmental Microorganisms Bank

Yong Jin Kim and Sang Seob Lee*

Research Center Kyonggi University

Korea Environmental Microorganisms Bank (KEMB) has been established as a microbial and genetic resource center for environmental industries. The KEMB plays an essential role as follows: 1-the collection and conservation of native environmental microorganisms and genetic resources, 2-the construction of systematic management system for effective conservation and application of microbiological resources for environmental industries, 3-the provision fundamental data for ecosystem research and microbial classification, and 4-the development of biological treatment system for bioremedation of environmental pollutant and ecosystem restoration.There are about 14,000 strains of bacteria collected from environments, at this time. These collections are classified in accordance with scientific and functional characteristics, respectively.It is considered to promote academic and industrial activities by supplying basic materials for research and industrial applications, which accomplish the ecological recovery through constructing eco-friendly bioremediation system by supplying basic microbial resources.

Keywords: Microorganism, Korea Environmental Microorganisms Bank (KEMB), Environmental Restoration, Bioremediation of Environmental Pollutant, Recovery of Ecosystem

Documents

· 2016. 5. 24. · Poster | 1 A001 Paenibacillus baekrokdamisoli sp., nov., Isolated from Soil of Crater