Poster

A001 A003 Paenibacillus baekrokdamisoli sp., nov., Isolated from Soil of Lentibacillus kimchii sp. nov., an Extremely Halophilic Crater Lake Bacterium Isolated from Kimchi

1 2 1 1 1 Keun Chul Lee1, Kwang Kyu Kim1, Jong-Shik Kim2, Dae-Shin Kim3, Young Joon Oh , Hae-Won Lee , Seul Ki Lim , Min-Sung Kwon , Jieun Lee , 1 1 3 Suk-Hyung Ko3, Seung-Hoon Yang3, and Jung-Sook Lee1,4* Ja-Young Jang , Jong Hee Lee , Hae Woong Park , Seong Woon Roh4, and Hak-Jong Choi1* 1KCTC/KRIBB, 2GIMB, 3World Heritage and Mt. Hallasan Research Institute, 4UST 1Microbiology and Functionality Research Group, World Institute of Kimchi 2 A novel bacterial strain Back-11T was isolated from sediment soil of crater Hygienic Safety and Analysis Center, World Institute of Kimchi 3 lake, Baekrokdam, Hallasan, Jeju, Republic of Korea. Cells of strain Back-11T Advanced Process Technology Research Group, World Institute of Kimchi 4 were Gram-staining-positive, motile, endospore-forming, rod-shaped and Biological Disaster Analysis Group, Korea Basic Science Institute oxidase- and -positive. It contained anteiso-C as the major fatty 15:0 A Gram-stain positive, aerobic, non-motile and extremely halophilic bacterium, acids, menaquinone-7 (MK-7) as the predominant isoprenoid quinone, designated strain K9T, was isolated from kimchi. The strain was observed diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine to be endospore-forming rod-shaped cells showing oxidase- and catalase- and four unidentified aminophospholipids as the main polar lipids, and positive reactions. Strain K9T was able to grow at 10.0–30.0% (w/v) NaCl meso-DAP as the diagnostic diamino acid in the cell-wall peptidoglycan. (optimum, 15.0–20.0%), pH 7.0–8.0 (optimum, pH 7.5) and 15–40°C (optimum, The DNA G+C content was 45.3 mol%. Phylogenetic analysis, based on 16S T T 30°C). The polar lipids of strain K9 consisted of phosphatidylglycerol, two rRNA gene sequencing, showed that strain Back-11 was most closely T unidentified phospholipids and an unidentified glycolipid. The predominant related to Paenibacillus taihuensis THMBG22 (95.5%) and fell into a clade isoprenoid quinone was menaquinone-7. The major cellular fatty acids in the genus Paenibacillus. On the basis of phylogenetic, chemotaxonomic T (>20% of the total) were anteisio-C and anteisio-C . The cell wall and phenotypic data, strain Back-11 represents a novel in the 15:0 17:0 peptidoglycan of strain K9T was determined as meso-diaminopimelic acid. genus Paenibacillus, for which the name Paenibacillus baekrokdamisoli sp. T T T The G+C content of genomic DNA was 48.2 mol%. Phylogenetic analysis nov. is proposed, with strain Back-11 (= KCTC 33723 = CECT 8890 ) as the based on the 16S rRNA genes revealed that the isolated strain was most type strain. 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.] A002 A004 Rhodanobacter aciditrophus sp. nov., an Acidophilic Study on the Mycelium and Morphological Characteristic of Bacterium Isolated from Mine Wastewater Naematoloma sublateritium

Hyeon-Woo Koh, Sudas Rani, and Soo-Je Park* Jongwoon Choi, Hasun Kim, Dongyong Shin, and Yunkyeong Lee* Department of Biology, Jeju National University Forest Research Institute of Gangwon-do

A novel strain (designated sjH1T), characterized as aerobic, Gram-stain This study showed to excellent mycelium growing at Glucose in circular negative, oxidase-positive, catalase-negative, motile and rod-shaped, was the carbon which was suitable for the third, mycelium growing of a N. isolated from mine wastewater. Phylogenetic analysis based on 16S rRNA sublateritium provisional results carbon circle regarding a nitrogen circle, gene sequences indicated that strain sjH1T belonged to the genus Rhodanobacter. and cultures of a N. sublateritium looked at malt extract in case of organic Strain sjH1T was closely related to Rhodanobacter thiooxydans LCS2T (98.0% nitrogen circles. However, a kind circular carbon nitrogen used to a test is 16S rRNA gene sequence similarity), Rhodanobacter denitrificans 2APBS1T restrictive, and provisional shall consist of various elements by a foundation. (97.7%), Rhodanobacter soli DCY45T (97.2%), and Rhodanobacter caeni This study looked in a phosphoric acid circle to affect the fourth, a MJ01T (97.0%). The DNA G+C content of strain sjH1T was 69.2 mol%. DNA-DNA mycelium of a N.sublateritium to growth and development. Also, If this relatedness (<60%) indicated that strain sjH1T represents a distinct species study added it was investigated p-aminobenzoic acid in a vitamin circle so that is separate from R. thiooxydans, R. denitrificans, R. soli, and R. caeni. that hypha growing was comparatively good. 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)]

www.msk.or.kr | 1 2016 International Meeting of the Microbiological Society of Korea

A005 A007 A Multiplex-PCR for Rapid Identification of 4 Enterococcus Flavihumibacter sediminis sp. nov., Isolated from Tidal Flat Species Using Species-Specific Primers from Comparative Sediment Genomics Do-Hoon Lee and Chang-Jun Cha* 1 2 2 2,3 2,3 Jongbin Park , Gwi-Deuk Jin , Yong Hyun Kim , Jae In Pak , and Eun Bae Kim * Department of Systems Biotechnology, Chung-Ang University 1Department of Animal Life System, College of Animal Life Sciences, Kangwon National University, 2Department of Animal Life Science, College of Animal Life A yellow-colored, Gram-stain-positive, aerobic, rod-shaped and non-motile Sciences, Kangwon National University, 3Division of Applied Animal Science, bacterial strain, designated AM3T, was isolated from the tidal flat in College of Animal Life Sciences, Kangwon National University 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 Enterococcus species are lactic acid frequently found in food/feed on 16S rRNA gene sequence similarity showed that strain AM3T belonged and human/animal guts. They are regarded as either commensal bacteria to the genus Flavihumibacter within the family and was or sometimes pathogens. For these reasons, rapid identification is required the most closely related to Flavihumibacter cheonanensis KACC 17467T in both food industries and clinical practices. Here, we investigated a new (98.3%), followed by Flavihumibacter solisilvae KACC 17917T (97.4%). rapid identification method based on species-specific genes from comparative DNA-DNA relatedness levels of strain AM3T were 30.6% to F. cheonanensis genomics for 4 Enterococcus species, 16S rRNA sequences of which are KACC 17467T and 42.6% to F. solisilvae KACC 17917T. The major isoprenoid similar. From genome comparison, we identified species-specific genes quinone was menaquinone (MK-7). The predominant polar lipids were for Enterococcus faecium, Enterococcus faecalis, Enterococcus durans and diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Enterococcus hirae, respectively. Four sets of primers were designed for The G+C content of the genomic DNA was 47.7 mol%. On the basic each species. After each primer set was tested and optimized for identification polyphasic taxonomic data, strain AM3T represents a novel species in the of corresponding species, the four primers were re-optimized for Multiplex- genus Flavihumibacter, for which name Flavihumibacter sediminis sp. nov. PCR using already known Enterococcus strains. This method was re-confirmed is proposed. 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 A008 Investigating the Inflammatory and Phenotypic Responses of Taxonomic Study of the Genus Hymenobacter Multiple Cultured Human Epithelial Cells to Predatory Bacteria Joo Won Kang, Mi Sun Kim, Ji Hee Lee, Seon Choi, Wasimul Bari and Ajay K. Monnappa* Da Hyun Kim, and Chi Nam Seong* Ulsan National Institute of Science and Technology Department of Biology, College of Life Science and Natural Resources, Sunchon National University Bdellovibrio-and-like-organisms (BALOs) are small Gram-negative predatory bacteria that attack and kill other Gram-negative bacteria including many The genus Hymenobacter, a member of the family Cytophagaceae (Stanier, pathogens. Although BALOs are considered to be a beguiling alternative to 1940), phylum , was proposed to group members producing antibiotics for treating multi-drug resistant infections, the possible large amounts of extracellular polymeric substances and spreading in thin, inflammatory and stress response in human cells to predatory bacteria pinkish layers on agar surfaces (Hirsch et al., 1999; Buczolits et al., 2006). remains to be explored. In this study, human intestinal (T84), alveolar At the time of writing, the genus comprises 36 species with Hymenobacter (NuLi-1) and breast (MCF10a) epithelial cells and the mouse Raw 264.7 roseosalivarius as a type species. The members of the genus Hymenobacter macrophage cell line were exposed to predatory bacteria, Bdellovibrio species have been isolated from a wide range of sources including air, soil, bacteriovorus HD100, B. bacteriovorus BY1 and Bacteriovorax stolpii EB1, fresh water, estuarine water, extreme conditions such as arid land, glacier, to determine if they elicit inflammatory responses. However, none of the uranium mine waste water treatment, pork, tidal flat and basal ice. human epithelial cells produced pro-inflammatory IL-8 and TNF-α or Common characteristics of Hymenobacter species are Gram-stain-negative, anti-inflammatory IL-6 and IL-10 cytokines in response to the predatory non-spore-forming, rods and producing large amounts of extracellular bacteria, whereas TNF-α was produced by the mouse macrophage cell polymeric substances (EPS). Major fatty acids were iso-C15:0, summed ω ω line. In contrast, exposure to a Gram-negative E. coli strain led to feature 3 (C16:1 7c and/or C16:1 6c) and summed feature 4 (iso-C17:1 I significantly higher amount of IL-8 and TNF-α from three human cell lines and/or anteiso-C17:1 B). The major respiratory quinone is menaquinone 7 and a mouse cell line, respectively. While, none of the predator strains (MK-7) and polyamine is sym-homospermidine. The G+C content of the were cytotoxic, lack of response was supported by confocal microscopy DNA varies between 53 and 70 mol%. results that showed the predatory bacterial strains did not bring about any [This research was supported by the project on survey and excavation of F-actin rearrangement in the mammalian cells. This in vitro study Korean indigenous species of the National Institute of Biological illustrates that predatory bacteria are not harmful to human epithelial Resources (NIBR) under the Ministry of Environment, Republic of Korea. It Ⅰ cells and adds valuable insight for future in vivo studies. was also supported by the CK(university for Creative Korea)- ]

2 | www.msk.or.kr Poster

A009 A011 OrthoANI: An Improved Algorithm and Software for Aquimarina 820-2 sp. nov., Isolated from Marine Sponge Calculating Average Nucleotide Identity Dysidea sp.

Imchang Lee1,2, Yeong Ouk Kim2,3, Sang-Cheol Park2,3, and Jongsik Chun1,2,3* Ga Eun Lee and Jin Sook Park* 1School of Biological Sciences, Seoul National University Department of Biological Science and Biotechnology, Hannam University 2Institute of Molecular Biology & Genetics, Seoul National University 3Interdisciplinary Program in Bioinformatics, Seoul National University An orange, rod-shaped bacterium, designated strain Aquimarina 820-2, was isolated from the marine sponge Dysidea sp., collected from Micronesia. Species demarcation in Bacteria and Archaea is mainly based on overall Cells were Gram-stain -negative, and catalase- and oxidase-positive. genome relatedness, which serves a framework for modern microbiology. Optimal growth was observed at 30°C, at pH 9, and with NaCl 3% (w/v). Current practice of obtaining these measures between two strains is The major fatty acids were iso-C15:0, iso-C17:0 3-OH, iso-C15:0 3-OH and shifting from experimentally determined similarity obtained by DNA-DNA iso-C15:0. Isoprenoid quinone was menaquinone. The DNA G+C content hybridization (DDH) to genome sequence-based similarity. Average nucleotide was 30.6 mol%. 16S rRNA gene sequence analysis showed that strain identity (ANI) is a simple algorithm that mimics DDH. Like DDH, ANI values Aquimarina 820-2 belonged to genus Aquimarina, the closest member between two genome sequences may be different from each other when being Aquimarina mytili PSC33T, with a gene sequence similarity of 96.8%. reciprocal calculations are compared. We compared 63,690 pairs of genome A number of phenotypic characteristics distinguished strain Aquimarina sequences and found that the differences in reciprocal ANI values are 820-2 from recognized members of the genus Aquimarina. On the basis of significantly high, showing over 1% in some cases. To resolve this problem data presented in this study, strain Aquimarina 820-2 is considered to of not being symmetrical, new algorithm, named OrthoANI, was developed represent a novel species of the genus Aquimarina. to accommodate the concept of orthology for which both genome sequences [Supported by NRF and Ministry of Oceans and Fisheries] 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 A012 A Bacterium Representing Novel Species in the Genus Acetobacter oryzifermentans sp. nov., Isolated from a Korea Sphingomonas, Isolated from Freshwater of Juam Reservoir Traditional Vinegar

Ji Hee Lee, Dae In Kim, Yong Seob Joo, Seo Young Kim, and Chi Nam Seong* Ga Youn Cho and Che Ok Jeon* Department of Biology, College of Life Science and Natural Resources, Sunchon Department of Life Science, Chung-Ang University National University An acetic acid bacterium, designated SLV-7T, showing great ethanol and A motile, aerobic and light yellow pigmented bacterium, designated strain acetate resistance was isolated from a Korean traditional vinegar. The cells 03SUJ6T was isolated from the freshwater of Juam reservoir, Republic of were Gram-staining-negative, catalase-positive, oxidase-negative, and non-motile Korea. Cells were Gram-stain-negative, catalase and oxidase activities are rods. The G+C content was 52.4 mol%. Strain SLV-7T was most closely negative. Strain 03SUJ6T grow at 15-37°C (optimally at 25-30°C) and at pH related to Acetobacter pasteurianus subsp. pasteurianus LMG 1262T, A. 6.0-8.0 (optimally at pH 7.0). Phylogenetic analysis based on 16S rRNA pasteurianus subsp. ascendens LMG 1590T, A. pasteurianus subsp. paradoxus gene sequences revealed that strain 03SUJ6T formed a distinct lineage LMG 1591T, and A. pomorum LHT 2458T with the 16S rRNA gene sequence within the genus Sphingomonas and was most closely related to Sphingomonas similarities of 99.9%, 99.8%, 99.7%, and 99.7%, respectively, but the DNA– daechungensis KCTC 23718T (97.44% 16S rRNA gene sequence similarity), DNA relatedness values between strain SLV-7T and the close related strains S. ginsengisoli KCTC 12630T (97.29%), S. humi KCTC 12341T (97.15%) and S. were 43.4 ± 8.6%, 21.8 ± 3.6%, 27.9 ± 5.7% and 25.2 ± 4.5%, respectively. rosea KCTC 12339T (97.01%). The DNA-DNA relatedness ratios were lower Phylogenetic analysis using the concatenated sequences of dnaK, groEL than 70%. The predominant fatty acids are C16:1, C17:1 ω6c, Summed feature 3 and rpoB indicated that the isolate formed a cluster with A. pasteurianus (C16:1 ω7c/C16:1 ω6c and/or C16:1 ω6c/C16:1 ω7c) and Summed feature 8 (C18:1 subsp. ascendens, but was distinct from other Acetobacter species. Average T ω7c and/or C18:1 ω6c). The major respiratory quinone was ubiquinone 10 nucleotide identity (ANI) values of strain SLV-7 with other genome sequenced (UK-10) and the major polyamine is sym-homo-spermidine. The DNA G+C Acetobacter species including closely related type strains were less than content of the genomic DNA was 64.7 mol%. On the basis of phenotypic, 90.52%. On the basis of genotypic, chemotaxonomic and molecular features, chemotaxonomic and phylogenetic data, strain 03SUJ6T can be considered strain SLV-7T clearly represents a novel species of the genus Acetobacter, as a novel species of the genus Sphingomonas. for which the name Acetobacter oryzifermentans sp. nov. is proposed. The [This research was supported by the project on survey and excavation of type strain is SLV-7T. Korean indigenous species of the National Institute of Biological Resources [This work was spported by the “Cooperative Research Program for Agriculture (NIBR) under the Ministry of Environment, Republic of Korea. It was also Science & Technology Development (Project No. PJ00999302)”, RDA] supported by the CK(university for Creative Korea)-I]

www.msk.or.kr | 3 2016 International Meeting of the Microbiological Society of Korea

A013 A015 Lapsobacter soli gen. nov., sp. nov., Isolated from Soil of a Thalassotalea litorea sp. nov., Isolated from Seashore Sand White Heron Nesting Site Heeyoung Kang, Haneul Kim, and Kiseong Joh* Min-Kyeong Kim1, Yujin Choi1, Tae-Su Kim1,2, Ji-Hye Han1,3, Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies Yochan Joung1,4, and Seung Bum Kim1* T 1Department of Microbiology and Molecular Biology, College of Bioscience and An aerobic, Gram-strain-negative, rod-shaped bacterium designated HMF4135 Biotechnology, Chungnam National University, 2Clinical Drug Manufacturing was isolated from a sand sample collected from the seashore of the East 3 Sea, Korea. Comparative 16S rRNA gene sequence analysis showed that Center, Osong Medical Innovation Foundation, Bacterial Resources Research T Team, Freshwater Bioresources Research Division, Nakdonggang National Institute strain HMF4135 belonged to the genus Thalassotalea and was most 4 closely related to Thalassotalea ponticola (96.4% sequence similarity). Strain of Biological Resources, Department of Biology, Inha University T HMF4135 was characterized by the predominance of C16:0, summed feature 3 (C ω7c and/or C ω6c), C ω9c and C 3-OH. The major polar lipids A Gram-stain-negative, motile by gliding, non-spore-forming and short 16:1 16:1 16:1 12:0 T were phosphatidylethanolamine and phosphatidylglycerol. DNA G+C content rod-shaped bacterial strain, designated R1-15 , was isolated from soil of a was 41.9 mol%. Based on the distinctive phenotypic characteristics and white heron nesting site (36° 21′ 52″ N, 127° 20′ 59″ E) and its taxonomic T T phylogenetic analysis, it is concluded that strain HMF4135 represents a position was evaluated using a polyphasic approach. Strain R1-15 grew at novel species of the genus Thalassotalea, for which the name Thalassotalea 15–37°C (optimum 30°C), at pH 6–7 (optimum pH 6) and in the presence of litorea sp. nov. is proposed. The type strain of the species is strain HMF4135T 0–1% (w/v) NaCl (optimum 0%) on 0.1× TSA. On the basis of 16S rRNA gene sequence (= KCTC 52154T = CECT -ingT). 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 A016 Flavobacterium keumense sp. nov., Isolated from Freshwater Roseovarius salarius sp. nov., Isolated from a Solar Saltern

Adaeze Ekwe, Joong-hyeon Ahn, and Seung Bum Kim* Haneul Kim1, Heeyoung Kang1, Yochan Joung2, and Kiseong Joh1* Department of Microbiology and Molecular Biology, Chungnam National University 1Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, 2Department of Biological Sciences, Inha University A yellow-pigmented, oxidase positive, catalase negative, Gram-negative, non-spore-forming, rod-shaped, aerobic, non-motile bacterial strain A Gram-staining negative and rod-shaped bacterial strain, designated designated K3R-10T was isolated from a freshwater source in Korea. The HMF2641T, was isolated from a solar saltern in Korea. A phylogenetic tree strain grew over a temperature range of 4oC to 35oC (optimum = 30oC) and based on 16S rRNA gene sequences showed that strain HMF2641T formed pH range 6-7 (optimum = 7). No growth was observed above 0.5% NaCl. a lineage within the genus Roseovarius. Strain HMF2641T was closely Phylogenetic analysis based on 16S rRNA gene sequence revealed that related to Roseovarius tolerans EL-172T (96.1% sequence similarity), R. strain K3R-10T belongs to the genus Flavobacterium and shares close azorensis SSW084T (95.7%) and R. mucosus DSM 17069T (94.7%). The similarities with Flavobacterium succinicans (97.02%), Flavobacterium major fatty acids of strain HMF2641T were summed feature 8 (comprising chungangense (96.39%), Flavobacterium branchiophilum (96.39%) and C18:1 ω7c and/or C18:1 ω6c), cyclo-C19:0 ω8c and C16:0. DNA G+C content of Flavobacterium piscis (96.32%). The polar lipid profile revealed the strain HMF2641T was 65.9 mol%. On the basis of the evidence presented presence of phosphatidylethanolamine, an unknown aminolipid and 3 in this study, strain HMF2641T represents a novel species of the genus unknown phospholipids. The DNA G+C content was 35.4mol%, MK-6 was Roseovarius, for which the name Roseovarius salarius sp. nov., is proposed the major quinone and homospermidine was the predominant polyamine. the type strain HMF2641T (=KACC 18421T =CECT 8857T). 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).

4 | www.msk.or.kr Poster

A017 A019 Zeaxanthinibacter aestuarii sp. nov., Isolated from Estuary A New Record of Didymella pinodella Isolated from a Fruit of Sediment and Emended Description of the Genus Asker2007 Red Pepper in Korea

Yun Hee Lee , Hye Im Jeong, Sang Eun Jeong and Che Ok Jeon* Tham Thi Duong and Hyang Burm Lee* Department of Life Science, Chung-Ang University Division of Food Technology, Biotechnology & Agrochemistry, College of Agriculture & Life Sciences, Chonnam National University A novel Gram-staining-negative, strictly aerobic, non-motile bacterium and yellow pigmented bacterium, designated strain S2-22T, was isolated A fungal strain EML-RPF-3 was isolated from a fruit of red pepper (Capsicum from an estuary sediment in South Korea. Cells of strain S2-22T were oxidase- annuum L.) in Korea. Pycnidia was solitary, formed on the agar surface or and catalase-positive rods without gliding motility. Growth was observed submerged, variable in shape and size, subglobose or elongated. Surface at 15–43°C (optimum, 37°C), at pH 5.5–9.0 (optimum, pH 6.5–7.5) and in of pycnidial wall was glabrous. A cirrhus on pycnidia was blackish and the presence of 0.0–10.0% (w/v) NaCl (optimum, 2.0%). The respiratory frequently observed on PDA medium. Pycnidiospores were hyaline, mostly quinone was only detected Menaquinone 6 (MK-6) and iso-C15:0, iso-C15:1 non-septate, sometimes 1-septate, ellipsoid, few ovoid, measured 2.0-3.2 G, iso-C17:0 3-OH and summed feature 3 (comprising C16:1 ω7c/C16:1 ω6c µm in diameter. Chlamydospores were dark brown, spherical to irregular, and/or C16:1 ω6c/C16:1 ω7c) were found out as the major fatty acids. The smooth to rough, terminal or intercalary and produced singly or in chains. major polar lipid was phosphatidylethanolamine and five unidentified BLASTn search of the rDNA ITS sequences via NCBI database indicated that aminolipids, an unidentified phospholipid, and three unidentified lipids the isolate matched Didymella pinodella (GenBank accession numbers, were also detected as the minor polar lipids. The G+C content of the KM030324 and KM030323) with identity values of 99.9% (992/993 bp) genomic DNA was 45.5 mol %. Phylogenetic analysis based on 16S rRNA and 99.9% (982/983 bp), respectively. Based on the morphological characteristics gene sequences showed that strain S2-22T formed a tight phyletic lineage and sequence analysis of the internal transcribed (ITS) regions, the isolate with Zeaxanthinibacter enoshimensis TD-ZE3T with a high bootstrap value was identified as Didymella pinodella belonging to Pleosporales. To our and their 16S rRNA gene sequence similarity was 94.6 %. Based on the knowledge, the species of Didymella pinodella represents a new record in phenotypic, chemotaxonomic and molecular features, strain S2-22T clearly Korea. 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 A020 A First Report of Pseudoalteromonas tetraodonis Isolated Pseudahrensia todarodis sp. nov., a Novel Bacterium from Apostichopus japonicas Guts Isolated from the Gut of a Japanese Flying Squid

Hyunjun Choi, Jihoon Jo, and Chungoo Park* Hyun Sik Kim, Pil Soo Kim, Dong-Wook Hyun, June-Young Lee, School of Biological Sciences and Technology, Chonnam National University Woorim Kang, Na-Ri Shin, Tae Woong Whon, and Jin-Woo Bae* Department of Life and Nanopharmaceutical Sciences and Department of Biology, More than 1,250 species of sea cucumber have been identified from the Kyung Hee University sea floor worldwide and approximately 20 of them are edible. Sea cucumbers represent commercially and medically valuable members of the group A novel Gram-stain-negative, non-motile, non-spore-forming, non-flagellated, Echinodermata, which itself is one of the most abundant and ecologically aerobic, beige-coloured and rod-shaped bacterium, designated strain KHS02T, successful marine invertebrate clades. One economically important species was isolated from the gut of a Japanese flying squid, Todarodes pacificus, as a source of seafood and traditional medicine is the sea cucumber collected from the East Sea, Korea. Phylogenetic analysis of 16S rRNA gene Apostichopus japonicus Selenka 1867, which is mainly found off the coasts sequences revealed that strain KHS02T formed a monophyletic clade with of northeast Asia. Moreover, salted sea cucumber intestines (know as P. aquimaris HDW-32T, with which it had the highest sequence similarity konowata in Japanese) have been considered as a gourmet and healthy (98.67%). Strain KHS02T grew optimally at pH 7 with 2% (w/v) NaCl at 25°C food. To explore whether there are beneficial microorganisms in sea on marine broth 2216, and could not grow without Na+. The predominant cucumber guts, we screened culturable microorganisms from guts of A. isoprenoid quinone was ubiquinone-10. The major fatty acids (more than japonicus, and isolated Alteromonas sp., Pseudoalteromonas sp., Vibrio 10% of total fatty acids) were summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω sp., and Shewanella sp.. Because it has been well known that Pseudoalteromonas 7c, 65.92%) and 11-methyl C18 : 1 ω7c (10.54%). The polar lipids of strain tetraodonis has antibacterial, bacteriolytic, agarolytic and algicidal activities, KHS02T comprised phosphatidylcholine, phosphatidylglycerol, phosphatidyle- we tested antibacterial activity using paper disc diffusion method about thanolamine, two unidentified aminolipids, an unidentified phospholipid our newly isolated Pseudoalteromonas tetraodonis CSB01KR in A. japonicus and an unidentified lipid. The genomic DNA G+C content was 58.6 mol%. guts. Carried out the antibacterial activity test against human pathogens, DNA-DNA hybridisation showed that the isolate shared 16.2% ± 1.3% the newly isolated Pseudoalteromonas tetraodonis CSB01KR has potent (reciprocal, 15.7% ± 2.8%) genomic relatedness with the type strain of the antibacterial activity. As a result, the Pseudoalteromonas tetraodonis CSB01KR closest species. In conclusion, we suggest this isolate is a novel species of may have considerable research value in medical and pharmaceutical fields. 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).

www.msk.or.kr | 5 2016 International Meeting of the Microbiological Society of Korea

A021 A023 A Novel Microbulbifer-like Bacteria Isolated from the Gut of Lacibacter nakdongensis sp. nov., Isolated from Sediments Purple Sea Urchin, Heliocidaris crassispina of Nakdong River

1,2 1,2 1,2 1,2 June-Young Lee , Pil Soo Kim , Dong-Wook Hyun , Hyun Sik Kim , Ji-Hye Han, Kiwoon Baek, and Mi-Hwa Lee* Na-Ri Shin1,2, Mi-Ja Jung1,2, Ji-Hyun Yun1,2, Min-Soo Kim1,2, 1,2 1,2 Bacterial Resources Research Team, Freshwater Bioresources Research Division, Tae Woong Whon , and Jin-Woo Bae * Nakdonggang National Institute of Biological Resources (NNIBR) 1Department of Life and Nanopharmaceutical Sciences, 2Department of Biology, Kyung Hee University A Gram-negative, non-spore-forming, orange pigmented bacterium, designated strain SS2-56T was isolated from the sediments of Nakdong River in Sangju-si, A novel Gram-negative, strictly aerobic bacterium, designated as strain T Gyeongsangbuk-do. Phylogenetic analysis based on 16S rRNA gene sequences AM134 , was isolated from the gut of a purple sea urchin Heliocidaris revealed that the isolate SS2-56T belongs to the family Chitinophagaceae, crassispina which was collected from the coastal waters of Dokdo, Korea. T and was most closely related to the type strains of Lacibacter daechungensis The 16S rRNA gene sequence analysis showed that strain AM134 H32-4T (96.64%) and Lacibacter cauensis NJ-8T (96.10%). Growth occurred belonged to the genus Microbulbifer in the family Alteromonadaceae and at 20-37°C (optimum temp. 30°C). The most abundant fatty acids in whole identified that the isolate had the highest sequence similarity with T T T cell of strain SS2-56 were iso-C16:0 (43.3%), iso-C17:0 3-OH (19.1%), summed Microbulbifer epialgicus F-104 (98.90% similarity). Strain AM134 grew feature 3 (C ω7c and/or C ω6c; 7.5%) and iso-C G (6.5%). The ˚ 16:1 16:1 15:1 optimally at 30 C, in the presence of 2% (w/v) NaCl and at pH 7. The G+C biochemical analyses using the API kit, Biolog and hydrolysis tests indicated content of genomic DNA was 56.1 mol%. The DNA-DNA hybridization that the strain could be clearly distinguished from related species of analysis showed the strain shared less than 27% genomic relatedness with T T Lacibacter. Based on the results of the polyphasic taxonomic analysis, a Microbulbifer epialgicus F-104 . The polar lipid profiles of strain AM134 novel species, Lacibacter nakdongensis sp. nov., is proposed to accommodate were constituted of phosphatidylethanolamine, phosphatidylserine, three this novel isolate. The type strain is SS2-56T. 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 A026 Flexivirga lutea sp. nov., an Actinobacterium Isolated from fontis sp. nov., Isolated from Freshwater the Stool of a Crested Ibis Gi Gyun Nam, Yochan Joung, and Jang-Cheon Cho* Woorim Kang, Dong-Wook Hyun, Pil Soo Kim, Na-Ri Shin, Hyun Sik Kim, Department of Biological Sciences, Inha University June-Young Lee, Euon Jung Tak, and Jin-Woo Bae* T Department of Life and Nanopharmaceutical Sciences and Department of Biology, A bacterial strain, designated IMCC1731 , was isolated from freshwater in Kyung Hee University 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 A novel Gram-staining-positive, aerobic, non-motile and coccus-shaped T belonged to the genus Emticicia within the family Cytophagaceae and was bacterium, designated strain TBS-100 , was isolated from the intestine of most closely related to Emticicia ginsengisoli Gsoil 085T (98.11% sequence a crested ibis, Nipponia nippon. The phylogenetic analysis based on the T T similarity) and DSM 17448 (94.67%). The DNA-DNA 16S rRNA gene sequences showed that the nearest species of TBS-100 T T T relatedness of strain IMCC1731 with respect to Emticicia ginsengisoli was Flexivirga alba DSM 24460 with 97.11% similarity and strain TBS-100 Gsoil 085T was less than 70%. The G+C content of strain IMCC1731T was belonged to the genus Flexivirga. The optimum growth condition for strain T 37.82 mol%. The predominant cellular fatty acids were summed feature 3 TBS-100 was 30°C, at a pH of 7 and 0% (w/v) NaCl. The primary cellular fatty T (C ω6c and/or C ω7c) and iso-C Based on the physiological, acids of strain TBS-100 were anteiso-C and iso-C . The predominant 16:1 16:1 15:0. 17:0 17:0 chemotaxonomic characteristics, DNA-DNA hybridization relatedness and isoprenoid quinone was MK-8 (H 70.2%) and MK-8 (H , 29.7%). The polar 4, 6 16S rRNA genes analysis, stain IMCC1731T is considered to represent a lipids were diphosphatidylglycerol, phosphatidylinositol, seven unidentified novel species of Emticicia, for which Emticicia fontis sp. nov. is proposed. lipid and an unidentified phospholipid. The whole cell-wall sugars of strain T T The type strain is IMCC1731 . TBS-100 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.]

6 | www.msk.or.kr Poster

A027 A029 Genomic Characteristics of Strain IMCC26207, a Flavobacterium inkyungensis sp. nov., Isolated from Non-colony-forming Actinobacterium, Isolated from an Freshwater of an Artificial Pond Oligotrophic Freshwater Lake Miri Park, Yochan Joung, Gi Gyun Nam, and Jang-Cheon Cho* Suhyun Kim, Ilnam Kang, and Jang-Cheon Cho* Department of Biological Sciences, Inha University Department of Biological Sciences, Inha University A Gram-staining-negative, non-motile, yellow pigmented bacterium, designated strain YMO21T, was isolated from an artificial pond (Inkyung Lake), Incheon. Although the phylum Actinobacteria is a common and numerically important T component in freshwater habitats, their potential roles in biogeochemical Growth of strain YMO21 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 process remain unknown due to the scarcity of reference genomic resources. T We report here the draft genome sequence of strain IMCC26207, a non- based on 16S rRNA gene sequences indicated that strain YMO21 belonged to the genus Flavobacterium and most closely related to Flavobacterium colony forming freshwater actinobacterium, with the description of the T genome properties. Strain IMCC26207 was isolated from the surface layer columnare IFO 15943 (97.8 % sequence similarity), Flavobacterium terrae R2A1-13T (97.2 %) and Flavobacterium vireti THG-SM1T (96.7%). DNA G+C of Lake Soyang by the dilution-to-extinction culturing method. Phylogenetic T analysis of 16S rRNA gene sequences indicated that strain IMCC26207 content of strain YMO21 was 32.1 mol%. The major fatty acids were formed a distinct lineage in the order Acidimicrobiales of the phylum 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 Actinobacteria. The closest relative among previously identified bacteria T was ‘Candidatus Microthrix parvicella’ (16S rRNA similarity: 91.7%). The lipid of strain YM021T was phosphatidylethanolamine (PE). Strain YM021 draft genome consisted of 10 contigs with a total size of 3.3 Mb and an showed low DNA–DNA relatedness with Flavobacterium columnare IFO 15943T (62.4 %). Based on the analysis of 16S rRNA gene sequences and average G+C content of 57.3%. The IMCC26207 genome was predicted to T contain 2,975 protein-coding genes and 51 RNA genes, including 45 tRNA biochemical characterization, strain YMO21 was considered to represent genes. Approximately 76% of the protein coding genes could be assigned a novel species in the genus Flavobacterium, for which the name Flavobacterium with a specific function. Annotation of the IMCC26207 genome showed inkyungensis sp. nov. is proposed. several traits of adaptation to living in oligotrophic freshwater environments. [Supported by grants from the Mid-Career Research Program through the Comparative analysis revealed that the IMCC26207 genome was distinct National Research Foundation (NRF) funded by the Ministry of Science, from ‘Candidatus Microthrix’ genomes; therefore, we propose the name ICT, and Future Planning] ‘Candidatus Limnosphaera aquatica’ for this bacterium. [Supported by a grant of the Mid-Career Research Program through the NRF]

A028 A030 Isolation of Three New Flavobacteriaceae Strains, Their Spirosoma aerophilum sp. nov., Isolated from Air Sample Genome Characteristics, and Proposal of the Name Aurantibacter yeongjongensis gen. nov., sp. nov Soo-Jin Kim, Jae-Hyung Ahn, Hang-Yeon Weon, Seung-Beom Hong, Soon-Ja Seok, Jeong-Seon Kim, and Soon-Wo Kwon* Yeonjung Lim1, Yochan Joung1, Seung-Jo Yang2, and Jang-Cheon Cho1* Agricultural Microbiology Division, National Institute of Agricultural Science, 1 2 Rural Development Administration Department of Biological Sciences, Inha University, National Marine Biodiversity Institute of Korea A rod-shaped, yellow-colored, Gram-stain-negative, non-flagellated, aerobic bacterium, designated as 5516J-17T, was isolated from an air sample Three Gram-stain-negative, non-motile, orange-colored bacterium, designated T collected from Jeju Island, Republic of Korea. It grew in the range of IMCC2192 , IMCC2184, and IMCC2644 were isolated from a marine solar 10-37°C (optimum 28-30°C), pH 6.0-11.0 (optimum, pH 7.0) and 0-1% saltern. Cellular growth occurred at 25-30℃, pH 6.0-7.5, and with 1-4% NaCl (w/v). Phylogenetic trees generated using 16S rRNA gene sequences (w/v) NaCl. The DNA G+C content of the strains were 35.9-36.0 mol%. The revealed that strain 5516J-17T belonged to the genus Spirosoma and major respiratory quinone was menaquionone-6 (MK-6) and predominant showed 96.9% sequence similarity to the most closely related species, cellular fatty acids were iso-C G, iso-C and iso-C 3OH. Major polar 15:1 15:0 17:0 Spirosoma linguale DSM 74T. The cellular fatty acids composed of large lipids were aminolipids, phospholipids, phosphatidylethanolamine. Phylogenetic amounts (>10% of total fatty acids) of summed feature 3 (C ω7c and/or analysis based on 16S rRNA gene sequence indicated that the strains 16:1 C ω6c) and C ω5c, and moderate amounts (5-10% of total fatty acids) belonged to the family Flavobacteriaceae and formed a distinct lineage 16:1 16:1 of iso-C 3-OH, iso-C and C . The DNA G+C content was 55.7 mol% that was independent of closely related genera: Winogradskyella (95.1-92.8%), 17:0 15:0 16:0 and MK-7 was the predominant isoprenoid quinone. Polar lipids were Flavirhabdus (94.2-94.3%), Bizionia (92.8-95.1%), and Corallibacter (93.9-94.0%). phosphatidylethanolamine, two unknown aminophospholipids, one unknown Average nucleotide identity (ANI) values (>98%) based on whole genome T aminolipid and one unknown lipid. On the basis of phenotypic and polyphasic sequence data (IMCC2192 and IMCC2644) and DNA-DNA relatedness T T study, it was suggested that strain 5516J-17 represent a novel (>90%) based on hybridization (IMCC2192 and IMCC2184) indicated that species within the genus Spirosoma, with the newly proposed name the strains belong to the same genomics species. The genome size of T T T Spirosoma aerophilum. The type strain is 5516J-17 (=KACC 17323 =DSM strains IMCC2192 and IMCC2644 were 2.9 Mb and 2.8 Mb, respectively, 28388T =JCM 19950T). 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]

www.msk.or.kr | 7 2016 International Meeting of the Microbiological Society of Korea

A031 A033 Lysobacter terricola sp. nov., Isolated from Greenhouse Soil Lacinutrix chionocetis sp. nov., Isolated from Gut of a Red Snow Crab Soo-Jin Kim, Jae-Hyung Ahn, Hang-Yeon Weon, Seung-Beom Hong, Soon-Ja Seok, Jeong-Seon Kim, and Soon-Wo Kwon* Hyangmi Kim1, Sunjoo Park2, Hyun-Woo Oh3, Agricultural Microbiology Division, National Institute of Agricultural Science, Kyung Sook Bae2, and Doo-Sang Park2,4* Rural Development Administration 1Freshwater Bioresources Culture Research Division, Nakdonggang National Institute of Biological Resources, 2 T Bicrobiological Resources Center, KRIBB A strain 5GH22-11 , which was isolated from greenhouse soil in Yangpyeong 3Core Facility Management Center, KRIBB, 4Department of Green Chemistry and region, Gyeonggi province, Republic of Korea, was characterized as aerobic, Environmental Biotechnology, UST Gram-stain-negative, flagellated, rod-shaped bacterium. The 16S rRNA gene sequence analysis showed that strain 5GH18-14T showed the highest T A Gram-negative, strictly aerobic, non-motile and rod-shaped bacterial sequence similarities with Lysobacter niabensis GH34-4 (98.6%), Lysobacter T T T strain, designated MAB-07 , was isolated from gut of a red snow crab. The yangpyeongensis GH19-3 (98.1%), Lysobacter fragariae THG-DN8.7 (97.9%), novel strain grew optimally at 25°C, at pH 7.0–8.0 and in the presence of Lysobacter terrae THG-A13T (97.3%), Lysobacter rhizosphaerae THG-DN8.3T T 3% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence (97.2%), Lysobacter tyrosinelyticus THG-DN8.2 (97.2%) and Lysobacter T T indicated that strain MAB-07 belongs to the type strains of species of the oryzae YC6269 (97.2%), revealing less than 95.5% sequence similarities genus Lacinutrix. Strain MAB-07T exhibited 16S rRNA gene sequence with all the other validly published species. Predominant quionone of T similarity values of 95.5-97.8% to the type strains of species of the genus strain 5GH18-14 was Q-8. Polar lipids were large amounts of phosphati- Lacinutrix. The predominant cellular fatty acids of strain MAB-07T were dylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and phos- iso-C15:1 G (27.5%) and iso-C15:0 (21.7%). The major respiratory quinine was phatidylmonomethylethanolamine, and moderate or small amounts of identified as MK-6. The polar lipids consisted of phosphatidylethanolamine, three unknown phospholipids and two unknown aminophospholipids. two unidentified phospholipids four unidentified aminolipids. The genomic DNA-DNA hybridization values with the closely related species were below DNA G+C content was determined to be 33.5% and its DNA-DNA relatedness 70%. DNA G+C content is 65.9 mol%. Based on the phylogenetic, physiological T values with the type strains of Lacinutrix venerupis, Lacinutrix mariniflava, and chemotaxonomic data, it was demonstrated that strain 5GH18-14 Lacinutrix jangbogonensis and Lacinutrix algicola were 28–35%. Based on represents a novel species of the genus Lysobacter, for which the name the data from this polyphasic taxonomic study, strain MAB-07T is considered Lysobacter terricola sp. nov. is proposed. The type strain is 5GH18-14T T T to represent a novel species of the genus Lacinutrix, for which the name (KACC 16954 =JCM 30862 ). Lacinutrix chionocetis sp. nov. is proposed. The type strain is MAB-07T (=KCTC 42767T).

A032 A034 Martelella suaedae sp. nov. and Martelella limoniae sp. Genomic Characterization of Strain IMCC26134, a Freshwater nov., Isolated from the Root of Halophytes Verrucomicrobial Strain Isolated from Lakewater

Eu Jin Chung1,2, Jung Moon Hwang1,2, Kyung Hyun Kim3, Ahyoung Choi1,2, Ilnam Kang1, Suhyun Kim1, and Jang-Cheon Cho1* 3 1 Che Ok Jeon , and Young Ryun Chung * 1Department of Biological Sciences, Inha University, 2Culture Techniques 1Division of Applied Life Science (BK21 Plus), Plant Molecular Biology & Biotechnology, Research Team, Nakdonggang National Institute of Biological Resources 2Freshwater Bioresources Culture Research Division, Nakdonggang National Institute of Biological Resources, 3Department of Life Science, Chung-Ang Strain IMCC26134, a freshwater verrucomicrobial strain, was isolated from University Lake Soyang by dilution-to-extinction culturing and formed small colonies on semisolid agar. IMCC26134 is an aerobic microorganism with optimal Two Gram-negative, aerobic and endophytic bacterial strains, designated growth at pH 7.0, at 20°C and salinity ≤ 0.2% NaCl. In phylogenetic YC7033T and YC7034T, were isolated from the roots of halophytes (Suaeda analyses, strain IMCC26134 constituted a robust branch within the family maritime and Limonium tetragonum) inhabiting in tidal flats of Namhae Opitutaceae of the phylum Verrucomicrobia. The closest relative of this Island, Korea, respectively. Strains YC7033T and YC7034T grew at 10–40°C strain was ‘Candidatus Diplosphaera colotermitum’ with a 16S rRNA gene (optimum at 28–30°C) and 4–40ºC (optimum at 28–30°C), respectively. sequences similarity of 92.6%. Here we describe the features of strain Strains YC7033T and YC7034T were able to grow at NaCl concentrations of IMCC26134, together with the complete genome sequence information 1–9% and 1–6%, respectively. The phylogenetic analyses of 16S rRNA gene and its annotation. The complete genome was comprised of one circular sequences showed that two strains were closely related to Martelella chromosome, with a length of 4,001,000 bp and a G+C content of 61.3%. endophytica YC6887T, Martelella mangrovi BM9-1T, Martelella radicis The IMCC26134 genome was predicated to contain 3,050 protein-coding BM5-7T and Martelella mediterranea DSM 17316T with 97.6–99.1% similarities genes and 56 genes for RNAs. The majority of the protein coding genes in 16S rRNA gene sequences. Sequence similarities with the type strains of (75.3%) were assigned a putative function. The genome of strain IMCC26134 another closely related genus, Rhizobium, were lower than 95.0%. Both revealed significant enrichment in genes encoding a wide spectrum of strains contained ubiquinone-10 (Q-10) as the major respiratory quinone glycoside hydrolases, sulfatases, polysaccharide lyases and carbohydrate system. The G+C contents of the genomic DNA of strains YC7033T and esterase, confirming that this organism was well equipped for the hydrolysis of YC7034T were 52.8 mol% and 62.2 mol%, respectively. The major fatty acids diverse polysaccharides. Strain IMCC26134 genome enhance understanding T T of both strains YC7033 and YC7034 were summed feature 8 (C18:1ω7c of the poorly known freshwater Verrucomicrobia phylum. 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.

8 | www.msk.or.kr Poster

A035 A037 Oleiagrimonas sediminis sp. nov., a Marine Bacterium Palleronia salinaecis sp. nov., a Halophilic Species Isolated Isolated from Tidal Flat Sediment and Emended Description from Solar Saltern of the Genus Oleiagrimonas Fang et al. 2015 and Oleiagrimonas soli Suhk Hwan Park and Geon Hyoung Lee* Department of Biology, Kunsan National University Sung-Hyun Yang1, Hyun-Seok Seo1, Chi Nam Seong2, and Kae Kyoung Kwon1* 1 A novel Gram-negative, strictly aerobic bacterial strain was isolated from Marine Biotechnology Research Center, Korea Institute of Ocean Science & T 2 solar saltern. This bacterial strain, designated as Kmb80 , was halophilic, Technology, Department of Biology, College of Life Science and Natural non-motile, non-spore forming, and rod-shaped. The colonies were 0.8-1 Resources, Sunchon National University mm in diameter, circular, pale-pink pigmented and with smooth edge. The strain was positive for oxidase and catalase activities. Growth occurred at A Gram-negative, aerobic, rod-shaped (1.4–3.6 ㎛ × 0.4–0.6 ㎛) and motile T pH 6.0–9.5 with optimal growth at pH 7.0, at temperatures of 20–40°C with marine bacterium, designated as MEBiC09124 was isolated from tidal flat optimal growth at 30°C, and in the presence of 0.5–20% (w/v) NaCl with sediment of the Sun cheon province, South Korea. The 16S rRNA gene T optimal growth at 5% (w/v). Based on the 16S rRNA gene sequence sequence analysis revealed that strain MEBiC08749 showed high similarity analysis, strain Kmb80T was assigned to the genus Palleronia within the T – with the Oleiagrimonas soli 3.5X (96.7%). Growth was observed at 18 family Rhodobacteraceae and was closely related to the type strain of – – 38°C (optimum 30°C), at pH 4.0 8.5 (optimum pH 7.5) and with 0 6% Palleronia soli with 96.9% of 16S rRNA gene sequence similarity. The (optimum 2.5%) NaCl. The predominant cellular fatty acids were iso-C15:0 T major cellular fatty acids of strain Kmb80 were C18:1 ω7c and/or C18:1 ω6c (22.6%), iso-C16:0 (16.0%), iso-C17:0 (10.0%) and summed feature 9 (comprised (38.8%) and C cyclo ω8c (30.4%). Ubiquinone 10 (U-10) was found to ω 19:0 of 10-methyl-C16:0 and/or iso-C17:1 9c; 22.5%). The DNA G+C contents is be the major respiratory quinone. The G+C content of the genomic DNA 66.1 mol%. The major respiratory quinone is Q-8. Several phenotypic was 67.3 mol%. On the based phenotypic properties clearly separated the characteristics such as production of acetoin and Enzyme activities of strain from other species of the genus. Accordingly, a new species of the Trypsin, α-chymotrypsin and N-acetyl-β-glucosaminidase differentiate strain T T genus Pallronia, Pallronia salinaecis sp. nov., is proposed (type strain MEBiC09124 from O. soli 3.5X . On the basis of this polyphasic taxonomic =Kmb80T =KCTC32522T = JCM19390T). 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 A038 Perlucidibaca aquatica sp. nov. Isolated from Freshwater Analysis of Non-ribosomal Peptide Synthetase Gene Cluster for Tolaasin Biosynthesis Kiwoon Baek, Ji-Hye Han, and Mi-Hwa Lee* Nakdonggang National Institute of Biological Resources 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 A Gram-staining-negative, non-motile, non-pigment, aerobic and rod-shape Sciences, Gyeongsang National University bacterium, designated BK-297T was isolated from small valley of the T Samcheok, Korea. Optimal growth of strain BK-297 was observed at 30°C, The whole sequence of non-ribosomal peptide synthetase(NRPS) gene pH 7.0 and in the presence of 0.5% (w/v) NaCl. Phylogenetic analysis T cluster was analysed. The gene cluster was composed of adenylation domains, based on 16S rRNA gene sequences revealed that strain BK-297 belonged condensation domains, and thiolation domains for 18 amino acids, consisting to the genus Perlucidibaca, forming a robust clade with member of the of tolassin, respectively. One thioesterase domain was found in terminal genus, and was most closely related to Perlucidibaca piscinae (97.8% region. Analysis of non-ribosomal peptide synthetase gene cluster for ω ω similarity). The major fatty acids were C16:1 7c and/or C16:1 6c (25.6%) tolassin biosynthesis. When the whole sequence of NRPS was compared and C16:0 (14.1%). On the basis of phyogentic analysis and genetic data, T to Pseudomonas tolaasii 6264 and Pseudomonas tolaasii NCPPB 2192, strain BK-297 represents a novel species of the genus Perlucidibaca, for there was not strong homology with these gene sequences from database. which the name Perlucidibaca aquatica sp. nov. is proposed. 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.

www.msk.or.kr | 9 2016 International Meeting of the Microbiological Society of Korea

A039 A041 The Diagnostic Characteristics are Highly Homoplasious Chthonobacter albigriseus gen. nov., sp. nov., Isolated from Used in Cladonia gracilis and Cladonia cornuta Grass-field Soil in Korea

Jae Eun So1,2, Soon Gyu Hong1,2, and Ji Hee Kim1* Do Hak Kim, Minsun Kim, Hansol Kim, Keunsoo Kang, and Tae-Young Ahn* 1Division of Polar Life Sciences, Korea Polar Research Institute Department of Microbiology, College of Natural Sciences, Dankook University 2Department of Polar Sciences, University of Science & Technology ED7T―a Gram-negative, non-motile, non-gliding, and aerobic bacterial The genus Cladonia usually lives on soil, moss and dead tree, and more strain―was isolated from grass-field soil in Cheonan, Korea. Strain ED7T than 400 species hitherto have been documented. Traditionally, the genus was able to grow at 20–42°C (optimum at 30–35°C), at pH 7.0–8.5 was divided into 7 taxonomic sections by morphology. However, the (optimum at pH 7.5–8.0), and at the absence of NaCl. According to taxonomical division is not well accord with that of molecular phylogenetic similarities of 16S rRNA gene sequences, strain ED7T was most closely research. The genus has a high morphological diversity, and the delimitation related to the genera Labrenzia (≤93.3%), Pleomorphomonas (≤93.1%), of many species has remained unresolved. The Cladonia gracilis group and and Prosthecomicrobium (≤93.1%). Phylogenetic analysis based on 16S allies in the section Cladonia shows an extreme phenotypic variety. rRNA gene sequence of strain ED7T revealed that it was affiliated with the Cladonia gracilis and Cladonia cornuta which are member of the C. gracilis order Rhizobiales, being most closely related to the genus Pleomorphomonas. group have been identified by variable characteristics. The present work Strain ED7T did not contain the nifH gene while its closest relatives, deals with the taxonomic problem between C. gracilis and C. cornuta by Pleomorphomonas koreensis, P. oryzae, and Prostehcomicrobium pneumaticum confirming the consistent phenotypic characteristics again with molecular did. The DNA G+C content of the genomic DNA was 71.7 mol%. The T phylogeny in species level. About 60 specimen were analyzed by 36 predominant fatty acids of strain ED7 were summed feature 8 (C18:1 ω7c morphological and chemical characteristics using the DNA sequences with and/or C18:1 ω6c), summed feature 2 (unknown fatty acid of 10.9525, C12:0 three loci (ITS rDNA, mtSSU and LSU rDNA). Specimen were collected from aldehyde, C14:0 3-OH and/or C16:1 iso I), C18:1 ω7c 11-methyl1, and C16:0 a few different regions covering bipolar areas and Korea. Phenotypic data 3-OH. The major isoprenoid quinone was ubiquinone 10 (Q-10). Based on were analyzed with several statistical methods. The study indicates that phenotypic, chemotaxonomic and genotypic characteristic data, the strain the consistent clade was not constructed in these species among the ED7T could be differentiated from other genus and considered that ED7T species level. The results show the unclear distinction between two species strain represents a novel species of a new genus in the order Rhizobiales, for secondary metabolites and geographical distribution. The characteristics for which the name Chthonobacter albigriseus gen. nov., sp. nov. is proposed. used for classical diagnosis may be highly homoplasious. The type strain is ED7T (=JCM 30603T =KCTC 42450T).

A040 A042 Zygomycete Fungi from Animal Dung in Korea Characterization and Description of Novel Strain RP18T, Isolated from the Forest Soil Thi Thuong Thuong Nguyen, Seo Hee Lee, Sarah Bae, Sun Jeong Jeon, and Hyang Burm Lee* Yongseok Ko, Han a Cho, Seoyoun Koo, and Tae-young Ahn* Division of Food Technology, Biotechnology and Agrochemistry, College of Department of Microbiology, College of Natural Sciences, Dankook University Agriculture & Life Sciences, Chonnam National University An aerobic, Gram negative, yellow-pigmented bacterium, strain RP18T was During a survey of zygomycota biodiversity in Korea, six isolates EML-DG8-1, isolated from the forest soil, Gwangju, Korea. The strain grew optimally on EML-DG-NH3-1, EML-UKD2-1, EML-UKD9-1, EML-UKD5-1, EML-UKD7-1 R2A medium at 30°C and in the absence of NaCl. The phylogenetic analysis were isolated from animal dung samples from June 2014 to Ferbuary based on 16S rRNA gene sequence suggested that the strain RP18T 2016. Sequence analysis by BLASTn search indicated that the isolates, belonings to the genus Sphingomonas, exhibiting the highest level of EML-DG8-1, EML-DG-NH3-1, EML-UKD2-1, EML-UKD9-1, EML-UKD5-1, sequence similarity with type strain of Sphingomonas kyeonggiense (95.96%), EML-UKD7-1 were closets to Absidia sp. (GenBank accession no. KC007314), Sphingomonas pituitosa (95.37%) and Sphingomonas jejuensis (94.71%). T Absidia glauca (GenBank accession no. AY944875), Mucor genevensis The major fatty acids of strain RP18 were, C14:0 2-OH,anteiso- C15:0, C16:0, (GenBank accession no. JN206044), Mucor hiemalis (GenBank accession C17:0 w6c, C18:1 w7c 11-methyl (>3% of the total fatty acids). Based on the no. JX045814), Mucor circinelloides (GenBank accession no. KT876701), evidence, The strain RP18T must be classified as a new species of the genus and Mucor mucedo (GenBank accession no. EU484199) with identity Sphingomonas. 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

10 | www.msk.or.kr Poster

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).]

www.msk.or.kr | 11 2016 International Meeting of the Microbiological Society of Korea

B001 B003 Occurrence of Viable, Red-pigmented Haloarchaea in the Comparative Analysis of Microbial Communities and Soil Plumage of Captive Flamingoes Organic Carbon Utilization Associated with the Depth and Thawing Effects on Tundra Soil in Alaska Seong Woon Roh and Jong-Soon Choi* Biological Disaster Analysis Group, Korea Basic Science Institute 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 Flamingoes (Phoenicopterus spp.) whose plumage displays elegant colors, inhabit warm regions close to the ocean throughout the world. The pink or In high-latitude regions, temperature has risen twice as fast as the global reddish color of their plumage originates from carotenoids ingested from average (0.3°C per decade) and this leads to the increase in microbial carotenoid-abundant food sources, since flamingoes are unable to synthesize degradability against soil organic carbon (SOC). Furthermore, the decomposed these compounds de novo. In this study, viable red-colored archaeal strains SOC is converted into green-house gases and their release could further classified as extremely halophilic archaea (i.e., haloarchaea) and belonging increase the rate of climate change. Thus, understanding the microbial to the genera Halococcus and Halogeometricum were isolated from the diversity and their functions linked with SOC degradation in soil-thawing plumage of flamingoes in captivity. Detailed analysis for haloarchaeal model is necessary. In this study, we divided SOC-rich soil from Alaska into community structure in flamingo feathers based on metagenomic data two depth regions (30−40 cm and 50−60 cm of depth) and incubated identified several haloarchaeal genera and unclassified sequences of the that for 108 days at 0°C. A total of 111,804 reads were obtained through class Halobacteria at the genus level. Carotenoid pigment analyses showed metagenomic study during the microcosm experiments, and 574−1,128 that a bacterioruberin precursor carotenoid in haloarchaea was identical of bacterial operational taxonomic units (OTUs) and 30−57 of archaeal to one of the pigments found in flamingo plumage. To the best of our OTUs were observed. Taxonomic analysis showed that the distribution of knowledge, this is the first report of viable extremophilic archaea in avian bacterial taxa was significantly different between two samples, while plumage, thus contributing to our understanding of the ecology of haloarchaea. archaea was similar. In detail, the relative abundance of phyla Actinobacteria The potential influence of haloarchaea as an environmental factor determining and largely increased in 30−40 cm and 50−60 cm of soil avian plumage coloration should be investigated in further studies. 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).]

B002 B004 Diversity and Distribution of Nitrite Oxidoreductase Alpha Metagenomic and Functional Analyses of the Consequences Subunit (nxrA) Gene in the Marine Sediments of Reduction of Bacterial Diversity on Soil Functions and Bioremediation in Diesel-contaminated Microcosms Rani Sundas, Hyeon-Woo Koh, and Soo-Je Park* Department of Biology, Jeju National University Jaejoon Jung1, Laurent Philippot2, and Woojun Park1* 1Department of Environmental Science and Ecological Engineering, Korea University Nitrite-oxidizing bacteria (NOB) are chemolithoautotrophs catalyzing the 2INRA Dijon, UMR 1347 Agroecologie, Dijon, France oxidation of nitrite to nitrate, which is the second step of aerobic nitrification. In marine systems, Nitrospina are major member as contribute to nitrogen The relationship between microbial biodiversity and soil function is an cycle. Still, two strains of the genus Nitrospina have been only isolated important issue in ecology, yet most studies have been performed in pristine from marine environment. Considering on their ecological contribution, ecosystem. Here, we assess the role of microbial diversity in ecological the investigations for their ecophysiology and environmental distribution function and remediation strategies in diesel-contaminated soils. Soil have been understudied due to fastidious cultivation and the insufficiency microbial diversity was manipulated using a removal by dilution approach of a functional gene marker. To estimate abundance, diversity and distribution and microbial functions were determined using both metagenomic analyses of Nitrospina, here we introduce nxrAencoding the alpha subunit of nitrite and enzymatic assays. A shift from - to Actinobacteria- oxidoreductase which used as functional and phylogenetic marker for that, dominant communities was observed when species diversity was reduced. and successfully applied into several marine sediments geographically Metagenomic analysis showed that a large proportion of functional gene distant location. We observed that Nitrospina diversities of the both polar categories were significantly altered by the reduction in biodiversity. The sediments were significantly lower than that of the non-polar samples. abundance of genes related to the nitrogen cycle was significantly reduced Moreover, nxrA-like sequences revealed an unexpected microdiversity in the low-diversity community, impairing denitrification. In contrast, the of Nitrospina with about 15000 identified operational taxonomic units efficiency of diesel biodegradation was increased in the low-diversity from six marine sediments. The nxrA gene copy numbers were ranged to 4 community and was further enhanced by addition of red clay as a stimulating 6.8 × 10 per gram of marine sediment sample. Conclusion, this study agent. Our results suggest that the relationship between microbial diversity estimated the distribution and diversity of Nitrospina and provided fundamental and ecological function involves trade-offs among ecological processes, information on the potential contribution to nitrogen cycle in marine and should not be generalized as a positive, neutral, or negative relationship. sediments. [Supported by grants from NRF (2015R1C1A1A01053750 & 2015M3D- 3A1A01064881)]

12 | www.msk.or.kr Poster

B005 B007 Effect of Oil Contamination on the Resilience of Taxonomic Cecal Microbiome from Broiler Chickens Differing in Body and Functional Structure in the Korean Tidal Flat Weight and Sex

Jaejin Lee1, Boram Kang2, and Tae Kwon Lee2* Kyu Chan Lee1, Dong Yong Kil2, and Woo Jun Sul1* 1Division of Life Sciences, Korea Polar Research Institute 1Department of Systems Biotechnology, Chung-Ang University 2Department of Environmental Engineering, Yonsei University 2Department of Animal Science and Technology, Chung-Ang University

A significant portion of crude oil from the oil shipping in the West Sea, Microbial community in ceca is involved in several important metabolisms Korea was transported to the tidal flat, where is an important habitat or such as water regulation, energy production during digestion and carbohydrate ecological zone due to high biological diversity. The objectives of this fermentations. To view the differences of microbial communities in the study were (i) to identify and characterize oil degrading bacteria, ii) to ceca, we analyzed chicken cecal microbiota based on bodyweights (high, characterize the recovery of taxonomic and functional structures over medium, low) and sex. In this study, 2×3 factorial experiment was designed time in Korean tidal flat. This study was conducted at Uihang-ri area, to predict bacteria positively related to host genotype and BW, using Taean, where chemical analysis revealed that total petroleum hydrocarbon Linear Discriminant Analysis Effect Size (LEfSe). Bodyweights and sex (C12 to C34) decreased from 3 μg/g to 0.5 μg/g over one year. Cycloclasticus, groups were clearly separated and statistically significant (p-value < 0.05). Alcalivorax, Pseudomonas and Thalassolituus were the predominant members In general, Actinomycetales members and Centipeda were positively related as oil degrading microorganisms during the crude oil degradation. Taxonomic to low BW while Enterococcus, unclassified Lactobacillales, unclassified (16S rRNA gene) and functional (aromatic ring hydroxylating dioxygenase) IncertaeSedis XIII and unclassified Ruminococcaceae were related to high community analysis revealed a distinct response to oil contamination by BW. In female chickens, several Firmicutes members (Sporobacter, Papillibacter, depth, but the initial community structure was recovered in both approches Unclassified Firmicutes) were mainly observed, positively relating to high over one year. The combination of 16S rRNA and dioxygenase gene analyses and low BW. In male chickens, on the other hand, many Firmicutes resulted in better understanding of the contributions to the crude oil members such as Coprococcus, Dorea and Roseburia were related to high removal of the indigenous microbial community and high degree of resilience BW rather than to medium and low BW. Since bacterial diversities abided of microbial community and diversity after the contamination. by BW and sex significantly differed, understanding of bacterial communities [Supported by Basic Science Research Program through the National deciphering chicken BW and health will help to improve breeding of Research Foundation of Korea (NRF) funded by the Ministry of Education, chickens in poultry industries. Science and Technology (NRF-2015R1D1A1A02061084)]

B006 B008 Bacterial Diversity and Species Composition in Three Structural Analysis of the Sensory Domain of the Endemic Baikalian Sponges Aromatic-responsive Transcriptional Activator PoxR from Ralstonia eutropha Eun-Young Seo1, Dawoon Jung1, Yochan Joung2, and Tae Seok Ahn1* 1Department of Environmental Science, Kangwon National University Vinod Vikas Patil1,2, Kwang-hyun Park1,2, Seung Goo Lee1,2, and Eui-Jeon Woo1,2* 2 Department of Biological Sciences, Inha University 1Korea Research Institute of Bioscience and Biotechnology, 2University of Science and Technology In Baikal, Russia, 3 species of sponges are living. Sponges are unique organisms with many species harboring symbiotic microbes that produce novel bioactive Phenol are considered as hazardous pollutants that pose threat to human compounds. To investigate bacterial diversity of Baikalian sponges, specimens life and surrounding ecosystem. Some bacteria are known to utilize of Lubomirskia baicalensis, Baikalospongia intermedia and Swartschewskia phenols as a carbon and energy source. PoxR is a transcriptional activator papyracea collected from Lake Baikal were processed by pyrosequencing. that control the metabolism of phenols. The sensory domain bind phenols We found differences in the species composition and diversity in bacteria and relays the signal to adjacent ATPase and DNA binding domains making among these sponges. Cyanobacteria accounted for the highest proportion it transcriptionally active that leads to transcription of several genes and second group was Proteobacteria in three sponges. The bacterial responsible for catabolism of phenol. Thus, it acts as a switch that can communities in B. intermedia and L. baicalensis were highly similar but control the expression of genes involved in catabolism of phenols. The less similar from the bacterial community associated with S. papyracea. structural information about the sensory domain has been elusive so far. Diversity of the bacterial community in S. papyracea was higher than in L. We have addressed the fundamental question on “How phenols are recognized baicalensis and B. intermedia. Especially, the relative abundance of inside bacterial cell” We coupled X-ray crystallography with isothermal Actinobacteria was higher in S. papyracea. Bacterial species in phyla titration calorimetry (ITC) to determine the structure of sensory domain Acidobacteria and Gemmatimonadetes were only found in S. papyracea. 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.

www.msk.or.kr | 13 2016 International Meeting of the Microbiological Society of Korea

B009 B011 Effect of Indigenous Microbiome in Soil on Salmonella Chromobacterium piscinae and Predation by Bdellovibrio enterica Survival bacteriovorus HD100

Suin Yang, Sora Kim, Jeong-A Lim, Jin-Woo Park, Wonsic Mun, Seong Yeol Choi, and Robert J. Mitchell* Jae-Gee Ryu, and Kyu Seok Jung* School of Life Science, Ulsan National Institute of Science and Technology Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration Bdellovibrio bacteriovorus HD100 is a predatory bacterium that attacks a wide range of Gram-negative bacterial pathogens. In this study, we found The objective of this study is investigation of the survival of Salmonella that Chromobacterium piscinae, a Gram-negative and violet-pigmented enterica, one of the foodborne pathogenic bacteria, in soil which has bacterium, is not predated by B. bacteriovorus when cultivated in Dilute indigenous microbial community. To do this when Salmonella enterica Nutrient Broth (DNB). However, C. piscinae can be predated on within a ATCC 13311 was introduced to soil where cherry tomato had grown for 10 nutrient-free buffer, HEPES. We examined and found that spent-DNB to 15 years in the green-house, survival of S. enterica were verified. S. media of C. piscinae significantly delays predation of E. coli MG1655 by B. enterica was inoculated to the autoclaved and non-autoclaved soil (8 log bacteriovorus HD100. Furthermore, the expression of the vioABCDE genes CFU/ml) and grown in green-house. The cell number of S. enterica within E. coli delayed predation of this prey. inoculated in autoclaved soil was maintained about 7-8 log CFU/ml until [Supported by grants from DARPA] 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 B012 Genomic Analysis of Confluentimicrobium naphthalenivorans Arbuscular Mycorrhizal Fungal Diversity in Post-mining Area NS6, a New Naphthalene Degrader and Natural Forest Area in Goesan, Korea

HyeIm Jeong, Kyung Hyun kim, and Che Ok Jeon* Hyeok Park1, Yu-Ra Bae1, Dong-Yeo Kim1, Kang-Hyun Ka2, and Ahn-Heum Eom1* Department of Life Science, Chung-Ang University 1Department of Biology Education, Korea National University of Education 2Division of Wood Chemistry & Microbiology, Korea Forest Research Institute Confluentimicrobium naphthalenivorans NS6, which was reported as a new novel naphthalene degrader isolated from a tidal flat (Int. J. Syst. Evol. Arbuscular mycorrhizal fungi (AMF) are one of the most widespread symbionts Microbiol., 2015, 4191-4195) was genome-sequenced for the better globally. AMF provide better nutrient absorptivity to host plant, enhance understanding of its naphthalene biodegradation properties. The complete tolerance against plant root pathogen, and also improve resistance against genome sequence of strain NS6 was characterized by a circular chromosomal soil contamination by heavy metal. Due to such benefits, AMF are very genome of 3.65-Mb with a 64.5% G+C content and three plasmids with important in harsh environments such as mines, deserts, or coastal areas. 184.7 kb, 157.3 kb, and 156.1 kb sizes. The genome analysis of strain NS6 In this study, we compared the difference of AM fungal diversity in post-mining showed that the genome had many dioxygenases that were probably area and natural forest area in Goesan, Chungcheongbuk-do, Korea. We related to the degradation of aromatic compounds and among them, four sampled 10 post-mining area soil samples, and 10 natural forest soil samples operons containing hydroxylating dioxygenase alpha subunit coding gene, with host plants. We extracted AMF spores in field soils, and identified a key enzyme in naphthalene degradation, could be candidates responsible spores using morphological characteristics and 18S rDNA sequence analysis. for naphthalene degradation and a phylogenetic tree based on Rieske Consequently, we discovered 5 species from 3 genera. Acaulospora was domain sequences of hydroxylase alpha subunits showed that the four the most abundant genus in post-mining area soils, and Ambispora was dioxygenase alpha subunits were respectively clustered into four different the most abundant genus in natural forest soils. These results show that clades. To investigate substrate specificities of the four putative naphthalene the AM fungal diversity of post-mining area soil is different to diversity of dioxygenase genes, four dioxygenase alpha subunit-encoding genes were natural forest soil. knock-outed using a double cross-over recombination approach and the [Supported by grants from NIFoS] 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.

14 | www.msk.or.kr Poster

B013 B015 Genome Analysis of Two Strains Belonging to the Genus Diversity of Bacteria Enriched with Coastal Area Samples Limnohabitans, a Major Bacterial Group in Keum River Contaminated with Petroleum Oils

Joong-hyeon Ahn and Seung Bum Kim* Seon-Hee Kim and Hyung-Yeel Kahng* Department of Microbiology and Molecular Biology, Chungnam National University Department of Environmental Education, Sunchon National University

From the previous investigation of freshwater microbiome in the four Coastal area samples contaminated with petroleum oils were collected major inland water bodies of Korea using pyrosequencing analysis, the and used for enrichment culture to isolate bacteria degrading petroleum genus Limnohabitans was found as a main bacterial group. In this study, oils such as gasoline, diesel, and kerosene. During enrichment culture for two strains affiliated with Limnohabitans were isolated, and designated a couple of months five hundred microliter samples were periodically 103DPR2 and 63ED37-2. These two strains showed nearly identical relationship taken per week and transferred to a sequential enrichment culture in with dominant groups of Limnohabitans appearing in different seasons as different media containing gasoline, diesel, or kerosene for a couple of determined by pyrosequencing. The complete genomes of two strains weeks. Analysis of bacteria enriched with different petroleum oils showed were analyzed to understand the properties of the strains as a dominant that bacterial diversity pattern from the diesel medium was similar to that group in the freshwater planktonic bacterial community. Genomic DNA from kerosene medium, but distinguished from that of gasoline medium. sequences of two strains were analyzed using single molecule realtime Three common representative bacterial genera from both diesel- and sequencing (SMRT) technique, and single, circular contigs were obtained kerosene-media were Pseudomonas, Maribacter and Rhodococcus. From for both strains. The sizes of two genomes were 2.95 Mbp (2,085 CDSs) the gasoline-media were only a couple of genera, Pseudomonas and and 3.37 Mbp (3,180 CDSs), the DNA G+C contents were 53.3 and 59.9 Alcanivorax found. Pseudomonas was the only genus obtained from all the mol%, the number of protein coding genes were 2,085 and 3,180, and the media with diesel, kerosene, and gasoline. A total of thirteen genera copies of rRNA genes were 6 and 9, respectively. The genome sequences isolated and identified from the enrichment culture using three kinds of were deposited to GenBank under the accession numbers CP011834 and petroleum oils were Alcanovorax, Algoriphagus, Brevundimonas, Demequina, CP011774. COGs related to amino acid transport and metabolism (category Erythrobacter, Hoeflea, Maribacter, Microbacterium, Phaeobacter, Pseudomonas, E), and energy production and conversion (category C) appeared as the Rhodococcus, Shewanella, and Thalassospira. most dominant metabolic categories for Limnohabitans genomes. This is [Suppported by grants from Ministry of Environment] the first report on the complete genomes of Limnohabitans, which will provide a basis for understanding their dominance in aquatic habitat.

B014 B016 Effects of Urea on Abundance and Community of Isolation and Characterization of Calcium Carbonate Nitrite-dependent Anaerobic Methane Oxidation Bacteria in Precipitating Bacillus and Sporosarcina Species from Concrete a Rice Paddy Hyun Jung Kim and Woojun Park* Sang Eun Jeong, Hyo Jung Lee, and Che Ok Jeon* Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University Department of Life Science, Chung-Ang University Microbially induced calcium carbonate precipitation (CCP) is a long-standing, To investigate the effects of urea on the abundance and community of but re-emerging environmental engineering process for production of nitrite-dependent anaerobic methane oxidation (n-damo) bacteria in a self-healing concrete, bioremediation, and long-term storage of CO . rice paddy, different amounts of urea (0, 45, 90, and 180 kg/ha) were 2 Three CCP-capable bacteria, Bacillus sp. JH3, Bacillus sp. JH7 and Sporosarcina applied three times (–1, 15, 45 days of transplantation) to a rice paddy and sp. HYO08 were isolated from two different concrete samples. Morphologically n-damo abundance and community were investigated using T-RFLP distinct extracellular CCP by the three strains were observed using energy (Terminal-Restriction Fragment Length Polymorphism) after 18 days of dispersive X-ray spectrometry mapping and field emission scanning electron third application. Soil cores were obtained and divided into three layers microscopy. Three strains showed different physiological characteristics: (top, 0–5 cm; middle, 10–15 cm; bottom, 20–25 cm). Abundance of n-damo urease activity, exopolysaccharides production, growth at alkali pH, and was not significantly different depending on the amount of urea application. high salt concentrations. Strain HYO08 had the highest urease activity and A phylogenetic tree of M. oxyfera-like bacteria based on 16S rRNA genes was more alkali-tolerant (pH 11). Strain JH7 was more halotolerant (5% derived from the rice paddy showed that M. oxyfera-like bacteria were NaCl) than two other strains. Barcoded pyrosequencing-based bacterial divided into four clades. Using an online program (Restriction Enzyme community analysis of the two concrete samples revealed low abundance Picker Online), NciI was selected as a suitable restriction enzyme to discriminate (<1%) of Bacillus and Sporosarcina species. High relative abundance of the n-damo clades. The n-damo based on T-RFLP patterns showed that Actinobacteria (<39%) and Cyanobacteria (<12%) indicated possibility of the clades b and c were the most abundant clades in all depths. In other novel CCP-capable bacteria present in concrete. Strain JH7 having particular, the relative abundance of clade c was higher along a depth both alkali- and halotolerant ureolytic activity was capable of plugging soil gradient in all rice paddies. However, the n-damo community was not pores and precipitating heavy metals. This study suggests the variance of different depending on the amount of urea application. CCP within different CCP-capable bacteria and the potential of utilizing [This study was supported by grants of the National Research Foundation isolated strains in environmental engineering fields. [Supported by grants of Korea (No. 2013R1A2A2A07068946, 2012R1A1B), funded by the Korean from NRF.] Government (MEST).]

www.msk.or.kr | 15 2016 International Meeting of the Microbiological Society of Korea

B017 B019 Genomic and Transcriptomic Analyses of Acinetobacter Screening of Highly Efficient Nitrogen Fixing Bacteria from oleivorans DR1 under Long-chain Alkane Soils and Their Utilization for Plant Growth Promotion

Chulwoo Park, Jaejoon Jung, and Woojun Park* Sun-hwan Jeong and Sang-Seob Lee* Laboratory of Molecular Environmental Microbiology, Department of Environmental Department of Life Science, Kyonggi University Science and Ecological Engineering, Korea University

The utilization of dinitrogen gas (N2) as a source of cell nitrogen is called Genomic analysis revealed the presence of two copies of each alkB-, almA-, nitrogen fixation. There are limited number of prokaryotes which can fix and ladA-type alkane hydroxylase (AH) in diesel-degrading Acinetobacter N2. The current was focused on the screening of highly efficient free living oleivorans DR1. DR1 cells could grow on C12–C30 alkanes, but not on C6–C10 nitrogen fixing bacteria from soil and their potential to promote the plant alkanes. Expression analysis of six AHs showed that alkB1 and alkB2 are growth. A soil sampling survey was conducted to collect the soil samples major AH-encoding genes under C12–C30 alkanes. Unlike other studies, our from Mun-kyeong and Yeon-cheon in South Korea. Initial screening of expression and mutant studies suggested that alkB-type enzymes could nitrogen fixing bacteria was conducted through the addition of mannitol be also responsible for lone-chain (LC) alkane degradation. Interestingly, and malic acid as carbon sources which help in the enrichment of such only almA2 was highly inducible among all AH genes under C30 while bacteria. Essential elements were also added in the growth medium which almA1 was constantly expressed. Expression of two ladA genes was not helps to fix dinitrogen gas (N2). Isolation and purification of diazotrophic observed under all tested conditions. RNA-seq analysis under C30 showed bacteria was done by dilution plate technique. Each strain was incubated up-regulated desaturases, suggesting changing of dynamic membrane for 5 days and their nitrogen fixation efficiency was measured by acetylene fluidity to facilitate adaptation under LC. Highly up-regulated trehalose reduction assay (ARA). In total 102 strains were measured by ARA and biosynthesis genes indicate possibility of trehalose lipid as a biosurfactant three bacteria namely, Cedeacea lapagei GTC 346T, Citrobacter youngae for LC alkne metabolism. Up-regulation of inorganic ion stress-induced CECT5335T and Enterobacter ludwigii EN-119T were showed to be highly operons such as pho, ssu, and especially kdp genes implies experience efficient and their nitrogen fixation efficiency was recorded up to 100%, inorganic ion-limited conditions probably due to difficulty in accessing 97.8% and 83.8%, respectively. Further research is focus on the identification charged ions. Phylogenetic and genomic analyses demonstrated that of optimal growth conditions such as O2 concentration, temperature, pH Acinetobacter is rare species possessing multiple alkane systems possibly and carbon source for high level of nitrogen fixation under filed condition through horizontal gene transfer. to promote plant growth. [This work was supported by grants from NRF]

B018 B020 Comparative Genomics Reveals Insights into Adaptation of Screening and Isolation of BTEX Degrading Bacteria from Ramlibacter Species to Different Soil Habitats Tongyeong Sea Water of Korea

Hyo Jung Lee and Che Ok Jeon* Hye Ji Kim and Sang-Seob Lee* Department of Life Science, Chung-Ang University Department of Life Science, College of Natural Science, Kyonggi University

Members of the genus Ramlibacter have been mainly isolated from various BTEX refers to the chemicals benzene, toluene, ethylbenzene and xylene. soil habitats. In particular, two Ramlibacter species, R. solisilvae 5-10T and They are the volatile components commonly associate with petroleum R. tataouinensis TTB310T, were isolated from evidently contrast soil products. BTEX can also dissolve in water and it may found in surface and habitats, forest and desert, respectively. Here, the complete genome of groundwater at contaminated sites. A survey was conducted to collect the strain 5-10 was sequenced and compared with strain TTB310. Strain 5-10 sea water and sediments samples from Tong-yeong region of South Korea harbors genes related to diverse metabolisms and stable mechanisms, (Masan gulf waste water treatment, Dot island, Jinhae gulf, and fish raising including ribosomal operon, posttranslational modification, protein turnover, farm). An enrichment culture was established using seawater to isolate chaperones (COG O), and amino acid and lipid transport and metabolism the BTEX degrading bacteria. A total of 85 bacterial strains were screened (COGs E and I), and cellular metabolisms, such as sugar, urea, and catechol which have capability to degrade BTEX. Of these 85 strains, 16 bacterial metabolisms and multidrug resistance genes in high abundances, which strains were identified which have high degradation efficiency. Out of may confer good fitness of strain 5-10 to forest soil. Whereas genes related these 16 strains, a strain designated FDM 14-2 showed 95.78% of benzene to response mechanisms and exopolysacharride biosynthesis genes, including degradation, 6.78% of toluene degradation 4.26% of ethylbenzene degradation transcription, signal transduction mechanisms, and carbohydrate transport and 0.62% of p-xylene degradation. Based on 16S rRNA gene sequence and metabolism (COGs K, T and G), and tolerance to oxidative stress, such analysis strain FDM 14-2 was identified as Bacillus aryabhattai B8W22T. 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.]

16 | www.msk.or.kr Poster

B021 B023 Development of Antifungal Compounds from Streptomyces Gut Microbiota of Mottled Skates, Raja pulchra from the sp. NF186 against Fusarium oxysporum f. sp. conglutinans Yellow Sea

Sung-Jin Cho1 and Sang-Seob Lee2* Eun Bae Kim 1Department of Biological engineering, Kyonggi University, 2Department of Life Department of Animal Life Science, College of Animal Life Sciences, Kangwon Science, College of Natural Science, Kyonggi University National University

Fusarium oxysporum is a soil borne plant pathogen in the class Hyphomycetes, Raja pulchra, Cham-hong-eo in Korean, is a kind of wild mottled skates. causes Fusarium wilt. Fusarium wilt caused by Fusarium oxysporum f. sp. Many Korean people have long enjoyed the unique stimulating flavor from conglutinans is a devasting soil-borne disease of cabbage plant in warmer skate ripening. Gut microbiota of the species has never been investigated climates that result in a sever reduction in yield. by using high-throughput sequencing before. Here, we investigated gut To search the efficient antagonistic bacteria against this pathogen, a soil microbiota of R. pulchra captured from the Yellow Sea in South Korea. Gut sampling survey was conducted to collect the samples from agricultural samples were collected from the large intestine of 6 individuals; 3 females land in South Korea. A dual culture assay was performed to screen the (F, 9.1±1.4 kg) and 3 males (M, 5.2±0.4 kg). To analyze microbial profiles, antagonistic bacteria against this pathogen. A total of 1159 bacterial 16S rRNA V4 region from gut contents was amplified by PCR and sequenced strains were screened for antagonistic activity. Out of these strains only 40 using the Illumina MiSeq Sequencer. At the phylum level, relative abundance of bacterial strains showed formation of clear zone around the pathogen Firmicutes (F : M = 51.7% : 40.4%, P=0.096) and Bacteroidetes (F : M = colony .Out of the forty strain only strain designated streptomyces NF186 28.4% : 15.1%, P=0.013) were higher in female skates, but Proteobacteria was selected for further analysis. was more abundant (F : M = 7.9% : 36.7%, P=0.0025) in males. At the genus Small subunit ribosomal RNA (16S rRNA) gene sequencing analysis identified level, Photobacterium (15.8%), Lactobacillus (13.6%), Prevotella (11.6%) were this strain as Streptomyces NF186 highest sequence similarity with Streptomyces the most abundant in the skate large intestine. The Photobacterium lactacystinicus OM 6519T. Optimization of the growth condition for the genus was much more frequent in males (F : M = 2.2% : 29.5%, P=0.011). production of high amount of antifungal compound from strain NF186 is These results indicate that there are differences between female and male under progress. 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]

B022 B024 Comparative Analysis of Bacterial Communities Isolated Glyoxylate Bypass Governs Bacterial Respiration under from Polychaete Habitat and Non-habitat in a Coastal Oxidative Stress Wetland Microcosm Sungeun Ahn and Woojun Park* Seyeon Shin and Hyung-Yeel Kahng* Laboratory of Molecular Environmental Microbiology, Department of Environmental Department of Environmental Education, Sunchon National University Science and Ecological Engineering, Korea University

Our previous study showed major bacterial species belonging to Bacillus, The glyoxylate shunt is an alternative to the TCA cycle that bypasses the Vibrio and Shewanella in polychaete, which was known to play an important carbon dioxide-producing steps and is essential for acetate and fatty acid role as a scavenger in coastal wetland. In order to investigate the impact of metabolism. The glyoxylate shunt is upregulated under conditions of ploychaete on the distribution pattern of bacterial communities, a coastal oxidative stress, antibiotic stress, and host infection, which implies that it wetland microcosm was constructed, in which polychaete was planted in plays important roles in stress defense and pathogenesis. In many some compartments of the mesocosm and incubated for a couple of bacterial species, including Pseudomonas aeruginosa, the isocitrate lyase weeks with the periodical addition of sterile feedstuff. Samples which (aceA) and malate synthase (glcB) genes are not in an operon, unlike in were taken up from the mesocosm were placed and spread on Marine Escherichia coli; thus, two genes might exhibit different transcriptional Agar (MA) media to obtain bacterial colonies. Eleven bacterial genera, Bacillus, responses to stress. Contrary to our expectations, deletion of aceA from P. Bartonella, Gaetbulibacter, Kocuria, Marinobacter, Oceanisphaera, Planomicrobium, aeruginosa improved cell growth under oxidative and antibiotic stress, Pseudomonas, and Vibrio were found from non-habitat of polychaete, while which could be attributed to decreased NADH production. Transcriptome from polychaete habitat were seventeen genera, Bacillus, Gaetbulibacter, data suggested that aceA mutants underwent a metabolic shift to aerobic Halobacillus, Idiomarina, Lysobacter, Marinobacter, Marinobacterium, denitrification, supported by additional evidence: upregulation of denitrification- Oceanisphaera, Planomicrobium, Pseudomonas, Rheinheimera, Rhodobacter, related genes, decreased oxygen consumption without ATP reduction, Shewanella, Sphingopyxis, Streptomyces, Tenacibaculum and Vibrio found. increased production of denitrification intermediates, NO and N2O, and Chemical analysis of the mesocosm wetland soils showed that soil composition increased cyanide resistance. A global bioinformatics analysis showed was changing during the life of polychaete planted. These findings suggested that only microorganisms capable of aerobic metabolism possess the that polychaete might make a great influence on change of wetland soil glyoxylate shunt. Our data suggest that the presence of isocitrate lyase, an environment as well as increased bacterial diversity in coastal wetland aerobic adaptation, appears to allow cells to bypass the TCA cycle, not mesocosm. only to preserve the carbon source, but also to tolerate oxidative stress. [Supported by Ministry of Food, Agriculture, Forestry and Fisheries] [Supported by NRF.]

www.msk.or.kr | 17 2016 International Meeting of the Microbiological Society of Korea

B025 B027 Endogenous Hydrogen Peroxide Increases Biofilm A Study on the Biodegradation of Petroleum Oils by Marine Formation by Inducing Exopolysaccharide Production in Microbial Consortia Acinetobacter oleivorans DR1 Seon-Hee Kim and Hyung-Yeel Kahng* In-Ae Jang and Woojun Park* Department of Environmental Education, Sunchon National University Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological, Engineering, Korea University A total of twenty one bacterial strains were isolated from crude oil- contaminated sand samples collected in Taean Malipo Beach. Three In this study, we investigated differentially expressed proteins in Acinetobacter bacterial strains among them, Brevibacterium frigoritolerans strain SHD-34, oleivorans cells during planktonic and biofilm growth by using 2-dimensional Pseudomonas brassicacearn subsp. neoaurantiaca strain SHG-4, and gel electrophoresis combined with matrix-assisted laser desorption time-of- Pseudomonas taeanensis strain SHG-7 were selected due to their faster flight mass spectrometry. We focused on the role of oxidative stress growth rate and used to construct four kinds of microbial consortia, resistance during biofilm formation using mutants defective in alkyl designated KⅡ-1(SHD-34, SHG-4), KⅡ-2(SHD-34, SHG-7), KⅡ-3(SHG-4, Ⅲ hydroperoxide reductase (AhpC) because its production in aged biofilms SHG-7), and K (SHD-34, SHG-4, SHG-7). Individual strains and consortia was enhanced compared to that in planktonic cells. Results obtained using were tested for their growth rate and biodegradation potential in a an ahpC promoter-gfp reporter vector showed that aged biofilms expressed minimal medium containing each 1% concentration of diesel, kerosene, or higher ahpC levels than planktonic cells at 48h. However, at 24h, ahpC gasoline for seven days' incubation, showing that SHG-4 of individual expression was higher in planktonic cells than in biofilms. Deletion of ahpC strains grew fastest in diesel, but growth rate of consortium KⅡ-3 was led to a severe growth defect in rich media that was not observed in faster than that of SHG-4 in diesel; in kerosene, KⅡ-3 grew fastest among minimal media and promoted early biofilm formation through increased the tested strains or consortia; in gasoline individual strain SHG-7 fastest production of exopolysaccharide (EPS) and EPS gene expression. Increased growth rate. In GC-MS analysis, KⅡ-1 exhibited biodegradation rates approximately 99% of the initial concentration of kerosene; KⅢ, lowest endogenous H2O2 production in the ahpC mutant in rich media enhanced biofilm formation, and this enhancement was not observed in the presence 56.8% biodegradation rate. In gasoline, all individual strains and microbial consortia showed 97-99.8% of the initial concentration. These facts of antioxidants. Exogenous addition of H2O2 promoted biofilm formation in wild type cells, which suggested that biofilm development is linked to suggested that consortium KⅡ-1 might perform an effective bioremediation of sea water and coastal wetland contaminated with petroleum oils. defense against H2O2. Collectively, our data showed that EPS production caused by H2O2 stress enhances biofilm formation in A. oleivorans. [Supported by NRF.]

B026 B028 Fungistatic Activity of an α-Aminophosphonate Chitosan Spatial Disturbances in Altered Mucosal and Luminal Gut Derivative against Aspergillus niger on Controlled Microgravity Viromes of Diet-induced Obese Mice

1 2 Yesupatham Sathishkumar , Kesavan Devarayan , Min-Soo Kim1 and Jin-Woo Bae2* 3 4 Byoung-Suhk Kim , and Yang Soo Lee * 1 2 Department of Biology, Department of Life and Nanopharmaceutical Sciences 1 Department of Forest Science and Technology, College of Agriculture and Life, and Department of Biology, Kyung Hee University 2Department of Basic Sciences, Tamil Nadu Fisheries University, Nagapattinam, 3 India, Department of BIN Convergence Technology, Chonbuk National University, Gut microbial biogeography is a key feature of host-microbe relationships. 4Department of Forest Science and Technology, College of Agriculture and Life Here, we conducted a metagenomic study to determine the composition Human space cruizing have been carrid sevearal decades since Apollo 11 of the mucosal and luminal viromes of the gut and to evaluate the impact landed on the Moon. Contamination by various living microorganisms of a Western diet on gut viral ecology. We found that mucosal and luminal within the international space station is ever increasing mainly due to viral assemblages comprised predominantly temperate phages. The mucosal human activity. In order to control of those of contaminants, microorganisms virome significantly differed from the luminal virome in low-fat diet-fed such as fungi and bacteria are important to maintain the well-being of the lean mice, where spatial variation correlated with bacterial microbiota astronauts during long-term living in aerospace since the immune functions from the mucosa and lumen. The mucosal and luminal viromes of high-fat, of astronauts are compromised under microgravity. And for the first time high-sucrose “Western” diet-fed obese mice were significantly enriched control of the growth of an opportunistic pathogen, Aspergillus niger, under with temperate phages of the Caudovirales order. Interestingly, this community microgravity is studied in the presence of α-aminophosphonate chitosan. alteration occurred to a greater extent in the mucosa than lumen, leading A low-shear modelled microgravity was used to mimic the conditions to loss of spatial differences; however, these changes recovered after similar to space. The results indicated that the α-aminophosphonate chitosan switching to a low-fat diet. Temperate phages enriched in the Western inhibited the fungal growth significantly under microgravity. In addition, the diet-induced obese mice were associated with the , Negativicutes inhibition mechanism of the modified chitosan was studied by UV-Visible and Bacteroidia classes, and temperate phages from the Bacteroidia class spectroscopy and cyclic voltammetry. This work highlighted the role of a particularly encoded stress- and niche-specific functions advantageous to bio-based chitosan derivative to act as a disinfectant in space stations to bacterial host adaptation. This study illustrates a biogeographic view of remove fungal contaminants. the gut virome and phage-bacterial host connections under the diet-induced [This work was supported by the Basic Science Research Program through microbial dysbiosis. the National Research Foundation of Korea (NRF) funded by the Ministry [Supported by grants from the Mid-Career Researcher Program, of Education (1301001076, 1401001209, 1201002578) and supported by NRF-2012-Forstering Core Leaders of the Future Basic Science Program] the Basic Research Laboratory Program (2014R1A4A1008140) through the Ministry of Science, Ministry of Trade, Industry & Energy (MOTIE).]

18 | www.msk.or.kr Poster

B029 B031 Isolation and Functional Classification of Marine Bacteria in Microbial Reduction of Nitrous Oxide under Environmentally Jeju Coastal Areas Useful for Industrial Purposes Relevant Concentrations

Seyeon Shin1, Dong-Heon Lee2, and Hyung-Yeel Kahng1* Doyoung Park and Sukhwan Yoon* 1Department of Environmental Education, Sunchon National University Korea Advanced Institute of Science and Technology 2Research Institute for Basic Sciences, Jeju National University

N2O has a global warming potential ~300 times that of CO2 and is recognized Hundreds of marine microorganisms were isolated from the waters of Jeju as the most potent ozone depletion agent. Many different pathways have coastal areas for the purpose of screening bacteria which can be used for been identified to contribute to production of N2O; however, the only the degradation of seaweeds. Two hundred bacteria were selected based identified biological sink of N2O is its reduction by Clade I and Clade II N2O on their enzyme activities detected by the experiment using a variety of reductases. A recent research suggested physiological difference between substrates. Thirty eight bacterial strains were found to exhibit protease activity; the organisms harboring two types of N2O reductases, especially in their Ten bacterial strains, amylase activity; eleven bacterial strains, cellulase affinity to N2O. To examine whether Clade I and Clade II organisms actually activity; sixty eight bacterial strains, phosphate solubilizing activity; twenty differ in their capability to consume and survive on trace N2O, the growth two bacterial strains, auxin formation activity; fifty one bacterial strains, of the two representative strains were compared under consistent supply siderophore producing activity. Furthermore, three hundred seventy five of trace N2O. A chemostat was designed to supply consistent flow of trace bacterial strains were additionally and independently tested for their (20 ppmv and 200 ppmv) N2O gas as the sole e- acceptor to Pseudomonas potential to degrade agar, carrageenan, alginic acid, and chitin which are stutzeri strain DCP-Ps1, and Dechloromonas aromatica strain RCB, while major components of seaweeds. It was found that one hundred nineteen acetate and NH4+ was continuously supplied with the liquid medium. bacteria strains had the ability to degrade agar, one hundred twenty three Unexpectedly, both organisms were able to attain steady-state in the bacterial strains, carrageenan, fifteen bacterial strains, alginic acid, and chemostat with the trace N2O (as low as 11.6 ppmv under steady-state) five bacterial strains, chitin. Interestingly, three bacterial strains among and did not differ significantly in their ability to consume trace N2O. qPCR them were found to be super-bacteria capable of degradation of agar, identified 3.3-fold difference in the cell numbers between the strains carrageenan, alginic acid, and chitin, suggesting that they may be strong when fed 20 ppmv N2O and that strain RCB exhibited 3.6-fold higher candidates used for a variety of industrial purposes in the future. per-cell N2O reduction than strain DCP-Ps1. These observations provide [Supported by Jeju Special Self-Governing Province] unprecedented insight into biological N2O reduction that may be omnipresent in the environment. [Supported by grants from NRF]

B030 B032 Isolation of Brackish Water Bacterioplankton from A Soil Bacterial Strain Displays Rifampicin Resistance by Saemangeum by High-Throughput-Culturing Method Based Inactivation of the Antibiotic on Cell-sorter Inoculation Ho-Jin Jang, Dae-Wi Kim, Do-Hoon Lee, and Chang-Jun Cha* Hyoung Tae Cheon, Yeonjung Lim, Suhyun Kim, and Jang-Cheon Cho* Department of Systems Biotechnology, Chung-Ang University Department of Biological Sciences, Inha University Rifampicin is an antibiotic used to treat bacterial infections. It is a key Cultivation of aquatic bacteria is necessary to understand the physiology component of anti-tuberculosis therapy, stemming from its inhibition of and ecological importance of diverse bacterial groups. High-Throughput- bacterial RNA polymerase. In the present study, we isolated a bacterial Culturing (HTC) based on dilution-to-extinction has been successfully strain from soil which was capable of transforming rifampicin. The strain applied to isolate representative bacterial groups. In this study, we applied had the 16S rRNA gene sequence similarity of 99.9% with Variovorax HTC to a brackish water sample collected from the just inside of the guangxiensis. The bacterium designated Variovorax sp. H1 also displayed Saemangeum reclamation wall. Inoculation of bacteria was performed resistance to rifampicin and was able to grow in R2A media supplemented μ using a FACS instrument. Bacterial cells were sorted into 9 fractions with rifampicin (100 g/ml). HPLC analysis showed that the strain completely according to forward and side scatter, and were inoculated directly from converted rifampicin to a metabolite within 3 days. Supernatant from the nozzle into low nutrient media, with a final cell density of 3 cells per 3-day-grown culture broth was also able to transform rifampicin, but well. After 7 weeks of incubation at 20°C, microbial growth was detected resting cells from the same culture did not. The metabolite was further in approximately 10% of 864 inoculated wells. Phylogenetic analyses of analyzed by LC-MS/MS and tentatively identified to be rifampicin quinone. 16S rRNA genes showed that FACS-HTC isolates were distributed in the Antibiotic susceptibility test was performed using a disk containing the diverse phyla/classes such as , , metabolite. Comparison of clear zone with that of rifampicin disk indicated , Bacteroidetes, Planctomycetes, Actinobacteria and that the metabolite lost its antibacterial activity. Our results showed that Verrucomicrobia, and included major bacterial groups representing either Variovorax sp. H1 isolated from soil inactivated rifampicin, conferring the marine or freshwater habitats. Particularly, an uncultured subgroup of antibiotic resistance. 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.]

www.msk.or.kr | 19 2016 International Meeting of the Microbiological Society of Korea

B033 B035 Specific Fungal Endophyte Resources: Diversity, A Physiologically, Genetically, and Morphologically Novel Characterization, and Comparative Analysis from Isolated Ammonia-oxidizing Archaeon, Nitrosocosmicus Contrasting Coastal Environments of Korea oleophilus, from Terrestrial Sediment

Young-Hyun You1 and Jong-Guk Kim2* Man-Young Jung1, Heeji Hong1, Eugene L. Madsen2, 1 1 1Marine Microorganism Team, National Marine Biodiversity Institute of Korea Md Arafat Islam , and Sung-Keun Rhee * 2 School of Life Science, Kyungpook National University 1Department of Microbiology, Chungbuk National University 2Department of Microbiology, Cornell University, Ithaca, New York, USA This study analyzed endophytic fungal distribution in three coastal environments of contrasting climatic, geographical, and geological characteristics: the volcanic Diverse ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota islands of Dokdo, the East Sea, and the West Sea of Korea. Fungal endophytes are thought to be responsible for ammonia oxidation across many terrestrial were characterized and analyzed with respect to the characteristics of habitats. In this study, we isolated and characterized an AOA, strain MY3, their coastal environments. Fungal endophytes were characterized and from coal tar-contaminated forest sediment. Strain MY3 is in clade analyzed with respect to the characteristics of their coastal environments. "Nitrosocosmicus", which is widespread and abundant in terrestrial habitats. For this purpose, common native halophyte communities from the three The cells of strain MY3 are large “walnut-like” cocci, divide by binary coastal regions were selected. Furthermore, isolates were characterized fission along a central cingulum, and have a tendency to form aggregates. by growth in specific salinity or pH gradients, with reference to previous Strain MY3 was mesophilic and neutrophilic. An assay of 13C-bicarbonate geographical, geological, and climate studies. Unlike East Sea or West Sea incorporation into archaeal lipid indicated that strain MY3 is capable of isolates, some of the Dokdo Islands isolates showed halophilic traits in strict autotrophy. The growth rates and yields of strain MY3 increased high salinity, and many could grow under extreme alkaline conditions. A when the growth medium was supplemented with several saccharides higher proportion of West Sea coast isolates showed halotolerance compared and complex organic compounds. The attachment of cells of strain MY3 to with the East Sea or Dokdo Islands isolates. These results suggest that XAD-7 hydrophobic beads and to adsorbent vermiculite demonstrated the unique fungal biota developed because of a close interaction between potential of strain MY3 to form biofilms, and with this attachment, the host halophytes and their specific environment, even within the same nitrification activity increased. The cell surface was confirmed as hydrophobic halophyte species. Therefore, this study proposes the application of by the extraction of strain MY3 from an aqueous medium with p-xylene. In specific fungal resources for the purpose of restoring sand dunes, salt- this study, to provide fundamental insights into the factors that shape the damaged agricultural land, or industrialization of halophytic plants. structure of AOA communities, key ecophysiological properties were [Supported by National Marine Biodiversity Institute Research Program established for a novel representative of the Nitrosocosmicus clade. (2016M00400).] [This research was supported by the Basic Science Research Program through the NRF (2014R1A1A2009901 and 2015R1A4A1041869).] B034 B036 Fish Possess Unique Microbial Communities Shaped by The Effect of pH on Nitrogen Dissimilation of Shewanella Surrounding Environments and Distinct from Other Vertebrate loihica PV-4 and Its Implication in N2O Emission and Nitrogen Retention Pil Soo Kim1, Jae Bong Lee2, Min-Soo Kim1, Tae Woong Whon1, 1 1 1 1 1 Dong-Wook Hyun , Ji-Hyun Yun , Na-Ri Shin , Mi-Ja Jung , and Jin-Woo Bae * Ha Yeon Kim and Suk Hwan Yoon* 1 Department of Life and Nanopharmaceutical Sciences and Department of Department of Civil and Environmental Engineering, Korea Advanced Institute of 2 Biology, Kyung Hee University, National Fisheries Research & Development Science and Technology Institute The use of N fertilizers in agricultural soils is the major contributer to Gut microbiota contribute to host organism by beneficial effects such as emission of N2O, a potent greenhouse gas. Loss of N from agricultural soils metabolism, immune response as well as ethological aspect. On the one due to denitrification is also a serious problem, as it causes an increased hand, host organisms also select their commensal microbiota that provides demand for N fertilizer and thereby, an increase in flux through the N advantages to survival or fitness of host. In recent papers, the composition cycle. Recent studies have suggested that pH may be a crucial factor in of gut microbiota is shaped by various factors including diets, habitat and regulation of N2O reduction and competition between denitrification (N phylogeny of the host. So far, the discoveries that suggested with host- loss) and respiratory ammonification (N retention). microbe interactions and co-evolutionary histories of vertebrate, yet unveiled Here, we have used Shewanella loihica strain PV-4 to investigate the in the gut microbiota of most of vertebrate species are uncharacterized. In simultaneous effects of pH on the ability of the organism as net N2O this study, we investigated bacterial communities of distal gut and luminal source or sink and competition between N-loss and N-retention. Strain - - contents from different habitats among more than 230 fishes, which PV-4 was incubated with 100 μmoles NO3 and 500 μmoles lactate as e collect across streams, lakes and sea. Bacterial metagenomic DNA from acceptor and donor, respectively and 44 μmoles N2O was added to 60-mL gut and luminal fluid of each fish species were sequenced by high-throughput headspace in 160-ml serum bottles with varying pH. At pH 6.0, transient + next generation sequencing. Network-based analysis and beta diversity accumulation of N2O up to 88 μmoles/bottle but no NH4 accumulation - plots based on Unifrac distances show salinity of habitats influences was observed after NO3 was completely reduced. At pH 7.0 and 8.0, no bacterial communities of fish. Additionally, fish gut microbiota clustered transient N2O accumulation was observed and strain PV-4 served as N2O differently with other vertebrate gut microbiota (mammals, reptiles and sink and respiratory ammonification activity was upregulated under the + birds). Thus, this study using culture-independent technology suggests high-pH conditions (300 and 1000 μmoles NH4 were recovered from - deep knowledge to understand unrevealed host-gut microbe interaction reduction of NO3 at pH 7 and pH 8, respectively). These results suggest in fish and surrounding environments, which can provides circumstantial that N2O emission reduction and N retention may be able to be simultaneously clue for trends of co-evolution in vertebrate and commensal microbes. achieved by controlling pH in the agricultural fields. [Supported by grants from KRF and NFRDI] [Supported by grants from KETEP]

20 | www.msk.or.kr Poster

B037 B039 Physiological Properties of Bacterioplankton during Analysis of the Rhizosphere Microbiome of Tomato Phaeocystis Bloom in Polynya of Amundsen Sea, Western Cultivars that are Resistant or Susceptible to Bacterial Wilt Antarctica Min-Jung Kwak1, Su Yeon Choi2, Ju Yeon Song1, Hyun Gi Kong2, 2 2 1 So-Jeong Kim1, Jong-Geol Kim1, Joo-Han Gwak1, Hee-Ji Hong1, Hyoung Ju Lee , Seon-Woo Lee , and Jihyun F. Kim * Woon-Jong Yu1, Md. Arafat Islam1, Soo-Je Park2, and Sung-Keun Rhee1* 1Department of Systems Biology, Yonsei University, 2Department of Applied 1Department of Microbiology, Chungbuk National University, 2Department of Biology, Dong-A University Biology, Jeju National University Bacterial wilt is a severe plant disease caused by the soil-borne bacterium Polynyas, areas of open water surrounded by sea ice, are sites of intense Ralstonia solanacearum. Recently, we initiated a whole metagenomic primary production and ecological hotspots in the Antarctic Ocean. We analysis of the rhizosphere communities of two tomato cultivars that are investigated the bacterioplankton response to phytoplankton bloom in resistant or susceptible to bacterial wilt. Hawaii 7996 is resistant to the Amundsen sea using metagenomic and metatranscriptomic analysis. disease, while Moneymaker is susceptible. 16S rDNA reads were extracted From metagenomics data, 12 representative bins were obtained based on from the whole metagenome data using blastn against the SILVA coverage plotting. At peak bloom, Polaribacter and Oceanospiriilales were database. Taxonomic analysis of the 16S rDNA sequences revealed that the dominant (30% and 10%, respectively) with high activity. However, most proportion of is higher in the rhizosphere of Hawaii7996 than of bins were 3–6% in the declining phase without predominant bins. in the rhizosphere of Moneymaker, whereas the proportion of Betaproteobacteria Polaribacter contributed mineralization of biopolymer. Most of LMW DOM is higher in Moneymaker than in Hawaii7996. Through the scaffold binning degradation is mediated by Gammaproteobacteria such as Oceanospirillales we have reconstructed an unknown Flavobacteriaceae genome named as (Ant4D3) genomes and SAR92. A comparative metagenomics and metat- FG1-H7996/R from the whole metagenome data of Hawaii 7996. Results ranscriptomic analysis of polynya bacterioplankton associated with different from the comparative genome analysis showed that FG1-H7996/R have phases of phytoplankton bloom enabled to provide insights into succession more genes associated with inorganic ion metabolism than closely related of bacteria niches of key bacterioplankton involved in carbon remineralization. Flavobacteriaceae species. These differences in the rhizosphere community [Supported by the Korea Polar Research Institute (KOPRI, PP16020).] 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)]

B038 B040 Bioprospecting for Amylase Producing Bacteria from Arctic Differential Compositions of Lichen Microbiomes in Sea Samples Cladonia gracilis According to the Positions at Thalli from King George Island, Antarctica Eungyeong Heo1, Haju Park2, Dockyu Kim2, and Eungbin Kim1* 1Department of Systems Biology, Yonsei University Hyun-Ju Noh1,2, Jang-Cheon Cho2, and Soon Gyu Hong1* 2 Division of Life Sciences, Korea Polar Research Institute 1Division of Polar Life Sciences, Korea Polar Research Institute, 2Department of Biological Sciences, Inha University The living organisms have adapted to this extreme conditions to survive. Generally, enzymatic efficiency is proportional to the temperature. However, Lichens are symbiotic organisms that are majorly composed of lichenized living organisms at sub-zero temperatures have different suitable temperature fungi (mycobiont) and green algae/or cyanobacteria (photobiont). However, for enzyme activities comparing with other organisms. lichen thalli also contain highly diverse microbes (microbionts) such as Polar bacteria can serve as an excellent source of cold-active hydrolytic bacteria, archaea, and fungi. Cladonia may provide different micro-environments amylases. As an effort to develop new bacterial resources for amylase, we to the microbial communities of Cladoia gracilis from King George Island, have screened a total of 495 biological samples from Chukchi Sea, for Antarctica. To reveal the microbial communities of different parts of the culturable bacteria with cold-active activities of extracellular amylases using lichenized fungi, we sectioned thalli from center, intermediate, and MB media containing starch as indicator substrates. The main condition marginal positions of colonies to apical, middle and basal parts. Since set for selecting the target bacteria was temperature. We selected the one apical parts are usually more exposed to sun light and wind, and has lower bacterium producing extracelluar amylase at 4°C and uninhabitable at humidity compared to basal parts, we considered this sectioning to 30°C. The target bacteria identified Pseudoalteromonas spp. by 16S rRNA represent different conditions. Bacterial 16S rRNA gene and eukaryotic sequence, named the bacteria Pseudoalteromonassp. strain EH1. nuclear large subunit (LSU) rRNA gene were analyzed by 454 pyrosequencing To purify the extracellular amylase, the medium was changed to Zobell method. Apical parts of thalli contained relatively simple bacterial communities, mediumwith 0.4% starch. After cultivation, the extracellular proteins over basal parts contained more complex with diverse phyla. LSU sequences 10 kDa size in supernatant were condensed by viva-flow and freeze dried. were mostly composed of diverse Cladonia genotypes. Algae and cyanobacteria The condensed proteins were separated by using ionic exchange column which are related in photosynthesis are represented in different parts of (GE Health care, q-column) and size exclusion column with FPLC. thalli. Although Cyanobacteria OTUs are enriched on basal parts, major [Supported by grants from KOPRI] 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.

www.msk.or.kr | 21 2016 International Meeting of the Microbiological Society of Korea

B041 B043 Measuring Patterns by Geographical Locations in Marine Diversity and Enzyme Activity of Penicillium Species Metagenome Data Using Newly Adopted Genotyping by Associated with Macroalgae in Jeju Island Sequencing Myung Soo Park1, Seobihn Lee1, Seung-Yoon Oh1, 2 1 Hoon-Je Seong1, Chung-Yeon Hwang2, Hong-Hee Won3, and Woo-Jun Sul1* Ga Youn Cho , and Young Woon Lim * 1 1Department of Systems Biotechnology, Chung-Ang University School of Biological Sciences, Seoul National University 2 2Division of Polar Biology and Ocean Sciences, Korea Polar Research Institute National Institute of Biological Resources, Environmental Research Complex 3Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA Several Penicillium species were isolated in a survey of extracellular As handling of the huge quantity of metagenomic data is difficult and enzyme-producing fungi from macroalgae along the coast of Jeju Island of time-consuming, we proposed more effective tool to analyze particular Korea. Penicillium species were identified based on morphological and β- sequences related to restriction enzyme sites for targeting reduced numbers tubulin sequence analyses. In addition, the halo-tolerance and enzyme of genes rather than to estimate whole genome sequences. In this study, activity of all strains were evaluated. A total of 28 strains of 19 Penicillium species whole genome shotgun sequencing method was compared to adapted were isolated. The diversity of Penicillium strains isolated from brown algae GBS procedure for verifying the efficiency. Additionally, single nucleotide was higher than the diversity of strains isolated from green and red algae. polymorphism’s differences were checked by functional categories. GBS– The common species were Penicillium antarcticum, P. bialowiezense, P. metagenome procedure was demonstrated with marine epipelagic samples brevicompactum, P. crustosum, P. oxalicum, P. rubens, P. sumatrense, and from 10 sites during the voyage from East Sea to Bering Sea. These samples P. terrigenum. While many strains showed endoglucanase, β-glucosidase, were processed by a restriction enzyme “ApeKI” and then sequenced by and protease activity, no alginase activity was detected. There was a Illumina HiSeq. In addition to environmental study, just 4 same site positive correlation between halo-tolerance and endoglucanase activity samples were sequenced by Illumina MiSeq for whole genome shotgun within Penicillium species. Penicillium has the potential to convert macroalgae sequencing to compare GBS method. Microbial pathway abundance to useful biomass. Among 19 Penicillium species, three species―P. kongii, patterns were compared between GBS method and general metagenomic P. olsonii, and P. viticola―have not been previously recorded in Korea. method. There were differences of entropy scores in metabolic genes [Supported by grants from the Marine BioResource Bank Program of the between East Sea and Western Pacific Ocean. Higher entropy values in Ministry of Ocean & Fisheries, Korea.] 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 B044 Comparative Metagenomic Analysis of Microbial Genome Reconstruction for Prediction of Metabolic Communities in Wheat Nuruk Fermentation Potential of Subsurface Archaea in Intertidal Mud Flat

1 1 2 2 Sediment Jeong-Ah Seo , Minjoo Kim , Ki Young Yoon , Min-Jung Kwak , 1 1 1 1 2 2 Joo-Han Gwak , So-Jeong Kim , Woon-Jong Yu , Md. Arafat Islam , Jihyun F. Kim , and Ju Yeon Song * Soo-Je Park2, and Sung-Keun Rhee1* 1 School of Systems Biomedical Science, Soongsil University 1 2 Department of Microbiology, Chungbuk National University Department of Systems Biology, Yonsei University 2Department of Biology, Jeju National University

Nuruk is a fermentation starter of Korean traditional turbid rice wines Subsurface in various marine and terrestrial anoxic environments harbors makgeolli and takju. As a natural fermentation starter, Nuruk is made with diverse archaea which are not cultivated yet. Although the function of grain flour and is fermented by various microorganisms such as fungi, those archaea are studied scarcely, heterotrophic lifestyle has been yeasts, and bacteria originated from environment. During fermentation, implicated based on organic carbon isotope tracer analysis. Intertidal zone microbial metabolism such as glycolysis could change biochemical conditions residing between terrestrial and marine habitats provides various niches of Nuruk. Since microbial composition of Nuruk has a key role in ripening for microbes. To investigate microbial community structure in mud flat rice wine, the quality of Makgeolli is mainly dependent on Nuruk. Nuruk sediment, pyrosequencing of 16S rRNA gene was performed using samples made of different wheat species were collected from each fermentation samples obtained below 2 m of the surface at intertidal zone at the step. A purification method of metagenomic DNA was established to Garolim bay. A total of 27 dominant phyla were identified. Archaea of exclude undesirable wheat genomic DNA and the extracted microbial Miscellaneous Creanarchaeotal group (MCG) was abundant with > 46% genomic DNA of Nuruk were subject to metagenomic sequence analysis. while Proteobacteria, Chloroflexi, Actinobacteria and Planctomycetes Microbial communities were monitored by the targeted amplicon sequencing were in the range of 6-15%. De novo assembly and binning of metagenomic of the 16s rRNA genes for bacteria and ITS regions for fungi. Dynamics of sequences from Illumina were performed for understanding the metabolic microbial structures in Nuruk could be characterized by fermentation time potential of subsurface archaea including MCG. Eight dominant archaeal and overall microbial communities of samples were decided by initial bins were obtained by coverage based binning to reconstruct genomes for inoculated microorganisms. 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]

22 | www.msk.or.kr Poster

B045 B047 Antibiotics Resistant Testing of Vibrio and Oxytetracycline Characterization of Ampicillin Resistant Aeromonas Species Resistant Bacteria Isolated from Fish Farming Water in Jeju Isolated from Domestic Streams

Son G. Nguyen, Mincheol Kim, Jungman Kim, Shalem Raj Padakandla, Yejin Jang, and Jong-Chan Chae* Nakwon Hwang, and Tatsuya Unno* Division of Biotechnology, Chonbuk National University Faculty of Biotechnology, College of Applied Life Science, SARI, Jeju 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 Olive flounder (Paralichthys olivaceus) is highly prized, common flatfish in from 28 locations in Korea. As a result of identification by 16S rRNA gene aquaculture in east Asian countries such as Korea and Japan. Several sequencing, the genus Aeromonas was predominant occupying 64.3% Vibrio species were reported as marine pathogenic bacteria. Due to the abundance among which Aeromonas veronii was dominant species. In intensive use of antibiotics feed additives in aquaculture, Vibrio and other order to identify the pathogenicity of the strains, PCR was employed to detect bacteria obtained antibiotics resistance genes and become potential risks aerolysin, serine protease, lipase, cytotoxic enterotoxin, and temperature-sensitive to humans. We isolated oxytetracycline resistant bacteria and Vibrio from protease. Only 0.17% of the strains contained tested five genes suggesting Olive flounder fish farms in Jeju island (includes sites: In, Discharge, and the low possibility of chronical infection by Aeromonas veronii inhabiting Sea coastal area near fish farms in triplication), then conducted antibiotic the natural environments. In susceptibility test against various antibiotics resistant test. The water samples were also tested by specific PCR for by disc diffusion assay, the antibiotic resistance of bacteria gradually detecting some potential pathogens (include Vibrio anguillarum, V. harveyi, strengthened from upstream to downstream of Han-river, representative and Streptococcus iniae). Total 288 Vibrio isolates were cultured on TCBS consecutive sampling location. This supports the anthropogenic effect on agar, and confirmed by genus specific PCR. Antimicrobial susceptibility antibiotics resistance in natural environments. 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 B048 Anthropogenic Effect on Prevalence of Antibiotic Resistance Genomic Insight of Bioplastic Production by Sphingobium in Natural Environments chungbukense DJ77T

Shalem Raj Padakandla1, Dae-Wi Kim2, Yejin Jang1, Woo Jun Sul2, Motakatla Venkateswer Reddy1, Young-Chang Kim2, and Jong-Chan Chae1* 2 1 Chang-Jun Cha , and Jong-Chan Chae * 1Division of Biotechnology, 2Department of Microbiology, Chungbuk National 1Division of Biotechnology, Chonbuk National University University 2Department of Systems Biotechnology, Chung-Ang University Sphingobium chungbukense DJ77T was isolated as biphenyl degrader and Although antibiotic resistant microorganisms are prevalent in natural capable of utilizing anthracene and phenanthrene as a sole carbon source. environments, the causing reason has not been elucidated clearly and The strain was originally identified as Sphingomonas chungbukensis and human activity is considered as one of important factors. In this study, the reclassified as Sphingobium chungbukense. The complete genome of S. prevalence of culturable antibiotic resistant bacteria and resistance-related chungbukense DJ77T was determined with Pacbio sequencing platform. genes were investigated from upstream to downstream of Han-river with The strain has two chromosomes (3.2 Mb and 1.1 Mb) and six plasmids ampicillin, lincomycin, tetracycline, kanamycin, erythromycin, cephalexin, (382 kb, 310 kb, 270 kb, 229 kb, 36 kb, and 19 kb) with 59.5 and 63.9% sulfamethoxazole, and ciprofloxin. The population of culturable antibiotic range of a G + C content. Poly-β-hydroxybutyrate (PHB) was produced resistant bacteria (ARB) was gradually increased from upstream to downstream. inside the cell of S. chungbukens using 4-methoxybenzoic acid as carbon While higher populations of ARB were appeared against ampicillin, licomycin, source. Electron microscopy confirmed the generation of granule inside kanamycin, and cephalexin, relatively lower abundances of ARB were detected the cell. Moreover, nitrogen limitation promoted PHB production and the against tetracycline, erythromycin, sulfamethoxazole, and ciprofloxin. physical properties of the produced PHB were analyzed. To better understand Antibiotics resistant genes (ARG) and mobile genetic elements (MGE) the mechanism in genetic level, omics analysis using the genome will be were also gradually increased in a number and amount from upstream to required. 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.

www.msk.or.kr | 23 2016 International Meeting of the Microbiological Society of Korea

B049 B051 Microbial Biogeography on the Sedimentary Environment Genome Sequence Analysis of the Soil Microbe Dokdonella Influenced by the Arctic Paleoclimate koreensis DS123

Dukki Han1, Seung-Il Nam2, and Hor-Gil Hur1* HyeonGwon Lee1, Min-Jung Kwak1, and Jihyun F. Kim1,2* 1School of Environmental Science and Engineering, Gwangju Institute of Science 1Department of Systems Biology and Division of Life Sciences, Yonsei University and Technology, 2Korea Polar Research Institute 2Strategic Initiative for Microbiomes in Agriculture and Food, Yonsei University

The Arctic marine environment consists of various microbial habitats that Isolated from the soil sampled near Dokdo island, Dokdonella koreensis harbor each local assemblage. The niche preference of microbial assemblages DS123 is an Gram-negative, motile, non-spore-forming and rod-shaped would directly or indirectly reflect the modern environmental change in that belongs to Xanthomonadaceae family. We obtained high quality the Arctic Ocean with oceanographic traits such as sea-ice dynamics, current sequence of 163-depth from PacBio HGAP3. Total bases of the genome circulation, and sedimentation. Such ecological niche for the Arctic microbial amount to 4,446,619 (70.3% G+C), which is predicted to encode 3,533 assemblages may provide putative evidences for geological events in CDSs, 60 transfer RNAs, and 2 ribosomal RNA operons. We annotated the response to the paleoclimate change. The present study describs a microbial gene functions by BLAST searches against the Uniref90, GenBank biogeography with well-dated high-resolution paleoceanographic records nonredundant, Pfam, COG, and KEGG databases. The 69 core genes are logged by a 5.4 m sediment core in the western Arctic Ocean. Distribution clustered in Xanthomonodaceae family and the genus Arenimonas is the of microbial populations was linked to extraordinary Holocene sedimentary nearest branch in the phylogenetic tree made with the core genes of the records, suggesting that microbial taxa may be indirectly available to the family Xanthomonadaceae. Functional categorization of the predicted reconstruction of paleoclimate. Indeed, glycerol dialkyl glycerol tetraethers genes shows that 72.85% of CDSs can be functionally assigned according (GDGTs), the archaeal lipid biomarker known as a specific indicator for to the COG category, and 30.77% of CDSs are assigned based on the Marine Group I Thaumarchaeota (MG-I) has been used for paleoceanography. Subsystem category. Many methylotroph sequences, expected to play However, we found the predominance of Marine Group II Euryarchaeota roles as the methanol oxidation, and mono-carbon metabolism, were (MG-II) rather than MG-I in the sediment layer (Unit C) containing found in DS123. The methanol-oxidation genes oxidate methanol into remarkably higher GDGTs, suggesting that MG-II contributes to the tetraether formaldehyde. In addition to, the genes related with twitching motility, lipid pool in sediments due to the input of terrigenous matter during the type 6 secretion system, CRISPR are found. 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 B052 Identification of a Variant of New Delhi Metallo-β- Genome Sequence of Maribacter dokdonensis DSW-8, a lactamase, NDM-9-Producing Klebsiella pneumoniae Marine Bacterium Isolated from Seawater of Dokdo, an in an Urban River in South Korea Island of the East Sea in Korea

Doris Y. W. Di1, Jeonghwan Jang2, and Hor-Gil Hur1* Jidam Lee1, Min-Jung Kwak1, Soon-Kyeong Kwon1, and Jihyun F. Kim1,2* 1School of Environmental Science and Engineering, Gwangju Institute of Science 1Department of Systems Biology and Division of Life Sciences, Yonsei University, and Technology, 2BioTechnology Institute, University of Minnesota, Saint Paul, 2Strategic Initiative for Microbiomes in Agriculture and Food, Yonsei University MN 55108, USA Maribacter dokdonensis DSW-8T, isolated from the seawater of Dokdo in Since the first report of the New Delhi metallo-β-lactamase (NDM-1) resistant the East Sea, Korea, is an aerobic rod-shaped Gram-negative bacteria Klebsiella pneumoniae in 2008, carbapenemase-producing Enterobacteriaceae belonging to Flavobacteriaceae. To analyze the genomic features of have been of great concern all over the world due to its multi-resistance Flavobacteriaceae bacterium isolated from seawater near the island, we encompassing nearly all antibiotics which refer to as ‘super bacteria’. In have determined the genome sequence of DSW-8 using Illumina 2014, a novel variant NDM-9 metallo-β-lactamase was identified from a sequencing platform. After read trimming, we have obtained 18,494,287 clinical K. pneumoniae PPH1303, which differed by a single amino acid high-quality reads and with 416-fold coverage of the genome size. After de substitution (E152K) from NDM-1. In our study, three K. pneumoniae (GJ1, novo assembly and gene prediction, 16 contigs with 4,434,543 bp (35.95% GJ2 and GJ3) isolated from Gwangju River were found to possess blaNDM-9 G+C content) were generated and 3,835 protein-coding sequences, 36 genes. Antimicrobial susceptibility test indicate resistance to aminoglycosides, transfer RNAs, and 6 ribosomal RNAs were detected. A phylogenetic tree carbapenems, cephems, folate pathway inhibitors, fosfomycin, and penicillins based on the 82 core genes among 181 sequenced members of the family but susceptible to fluoroquinolones, phenicols, tetracycline and miscellaneous Flavobacteriaceae showed that DSW-8 forms a sister clade with agents. Whole genome sequencing revealed that the IncFII(Y)-like plasmids Maribacter forsetii DSM 18668. In the genome of DSW-8, the genes carry blaNDM-9, aadA2, aph(3’)-Ia, sul1, and dfrA12 genes along with mercury encoding the proteins associated with gliding motility, molybdenum resistance operon are approximately 108-kb. blaLEN-13, blaTEM-1, and blaCTX-M-65, cofactor biosynthesis, and several kinds of carbohydrate utilization. ant(3’)-Ia, fosA, mph(A), aqxA, and oqxB genes were identified. Genes Moreover, the genes encoding TRAP-type C4-dicarboxylate transport encoding NDM-9 were located between insertion sequences IS15, IS26 system were detected as DSW-8 specific genes. Gene content of DSW-8 and IS15D1 upstream and ISVsa3, IntI1, and resolvase downstream on may provide the useful information in the carbon utilization of the marine plasmids. These mobile elements are predicted to be capable of site-specific flavobacteria. recombination, transposition and switching of integron cassettes which increase the potential of horizontal gene transfer among microorganisms in the environment.

24 | www.msk.or.kr Poster

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.

www.msk.or.kr | 25 2016 International Meeting of the Microbiological Society of Korea

C001 C003 3D Structure of Family 5 Extracellular Solute-binding Protein Secondary Metabolites for Plant Growth Promoting in Bifidobacterium longum KACC 91563 Produced by Rhodobacter capsulatus PS-2

Junsang Ham, Han-ha Chai, Hyoun Wook Kim, Ki Moon Bong1, Jong Min Kim1, In Cheol Park2, Bu Min Kim, and Mi-Hwa Oh* Chul Won Lee3, and Pyoung Il Kim1* National Institute of Animal Science, RDA 1Jeonnam Bioindustry Foundation, Bio Control Research Center 2Agriculture Microbiology Division, National Academy of Agricultural Science We found Bifidobacterium longum subsp. longum KACC 91563 alleviates 3Department of Chemistry, Chonnam National University food allergy in animal model, and examined ESBP (extracellular solute binding protein) in Bifidobacterium longum subsp. longum KACC 91563 Hormones of plant growth promoting (PGP), which are produced in small alleviates food allergy through mast cell suppression in a previous research. quantity, affect various activities in plant growth and development. PGP We tried to understand 3D structure of the ESBP. Protein family, domain, was the effect on crops productivity in fields of agricultural industry. There and biological process of ESBP in Bifidobacterium longum subsp. longum are several types of auxin (indole-3-acetic acid; IAA), gibberellin (GA3) and KACC 91563 were compared to that of Bifidobacterium longum subsp. abscisic acid (ABA). In this study, Rhodobacter capsulatus PS-2 strain was longum JDM 301, but partial similarity was found. 3D structure of ESBP in isolated from paddy soil. The growth rate and PGP production of R. capsulatus Bifidobacterium longum subsp. longum KACC 91563 was predicted on the PS-2 were investigated under different culture conditions. To measure the basis of the ESBP in Salmonella enterica subsp. enterica serovar Typhimurium productivity of PGPs, LC-MS/MS method was performed in the multiple which has 40.4% similarity. ESBP in Bifidobacterium longum subsp. longum reaction monitoring (MRM) modes. MRM was employed for qualitative KACC 91563 may bind to a ligand peptide (Lys-Leu-Lys) weaker than that measurement. The MRM transitions were monitored as 176→130 for IAA, of Salmonella enterica subsp. enterica serovar Typhimurium. Allergy 265→148 for ABA and 347→238 GA3, respectively. The results showed alleviation could be a strain-specific characteristic of beneficial microbes. that the R. capsulatus PS-2 produced PGP, IAA, GA3 and ABA for plant growth promoting. [Supported by grant from RDA.]

C002 C004 Genetic Strategies for Pikromycin Over-production in Cultivation Conditions for Mass Production of an Antifungal Streptomyces venezuelae Lipopeptide from Bacillus methylotrophicus GH1-13

Joon-Sun Choi, Ji-Eun Kim, and Jung-Hye Roe* Jong Min Kim1, Ki Moon Bong1, Gong Min Kim1, JaeKyeong Song2, 3 1 School of Biological Sciences, College of Natural Science, Seoul National University Chul Won Lee , and Pyoung Il Kim * 1Jeonnam Bioindustry Foundation, Bio Control Research Center Pikromycin is the 14-ring macrolide antibiotic, produced by type 1 PKS 2Agriculture Microbiology Division, National Academy of Agricultural Science system in Streptomyces venezuelae ATCC15439. It is known as a potential 3Department of Chemistry, Chonnam National University raw material for ketolide compounds. For pikromycin over-production, we established host engineering strategies which include promoter change In this study, optimal culture condition for the mass production of antifungal and removal of unnecessary genes. As the first step, we examined the time lipopeptide producing microbe was achieved. Microorganism was kindly course of pikromycin production in submerged culture condition. Pikromycin provided by Korean Agriculture Culture Collection (KACC), Republic of began to appear from the beginning of the stationary phase and accumulated Korea. The optimal culture conditions such as pH, inoculum volume and throughout the whole culture period. For over-expression of the pikromycin medium composition were established. Medium composition was selected biosynthetic gene cluster during the stationary phase, we selected several by the concentration gradients of yeast extract, glucose, NaCl, K2HPO4, promoter candidates exhibiting strong activities after late exponential Na2CO3, MgSO4. After mass cultivation, cell-free supernatant was used to phase. We identified transcriptional activities of promoter candidates by assay antifungal activity using the paper disc method and the antifungal using GUS (β-glucuronidase) reporter system and S1 nuclease mapping lipopeptides were identified by LC-MS/MS (SCIEX API 2000 series). Antifungal assay. Through transcriptome analysis of S. venezuelae ATCC15439, we lipopeptides of molecular weight 1044.9 Da, 1486.3 Da and 1506 Da were also selected highly expressed gene clusters for putative secondary metabolites. analyzed. From the generated protonated ion peaks in LC-MS/MS, 1044.9 Using homologous recombination, we deleted three gene clusters predicted m/z and 1486.3 m/z, 1506.0 m/z were identified as surfactin and fengycin, to encode polyketide biosynthetic enzymes. This triple mutant produced respectively. higher level of pikromycin than the wild-type. [Supported by grants from RDA]

26 | www.msk.or.kr Poster

C005 C007 Multi-drug Resistant Staphylococcus aureus Growth Growth Inhibition of Multidrug Resistant Staphylococcus Inhibition by Violacein Produced by Pseudoduganella aureus Using Cotton Fabric with Violacein Derivative Natural Isolated Strain NI28 SeongYeol Choi1, HeeUn Kwon1, SooYeon Kim1, YeongMi Kwon2, 2 3 1 SooYeon Kim, SeongYeol Chio, and Robert J. Mitchell* ChangSeok Lee , JinHyeng Lee , and Robert J. Mitchell * 1 Ulsan National Institute of Science and Technology School of Life Science, Ulsan National Institute of Science and Technology 2YeeJoo Research Institute, YeeJoo Corporation 3 Microorganisms are causing all kinds of human diseases varying from Korea Institute of Ceramic Engineering and Technology infections to cancer. Nowadays the overuse of antibiotics resulting in multi-drug resistant organism is becoming critical threats to health issues, Violacein is one of antibacterial pigment produced by various natural however, few solutions are found to fix this problem. For the development bacteria such as J. lividum, C. violaceum. It Inhibits gram positive bacterial of therapeutic material, therefore, we should consider both to cure growth and kills protozoans which consume bacteria in nature. Even this diseases by antibiotic development or rediscovery of traditional one as very powerful advantage, however, violacein and its derivatives are too well as not to build drug resistance. In this study, we introduce the hydrophobic to dissolve aqueous phase. This study focus on its antibacterial multi-use agent, violacein with variety of profitable effects such as anticancer, application. The violacein can block gram positive bacteria moreover it antibiotic, antifungal and antiprotozoan effect. The violacein is produced can block growth of methicillin resistant Staphylococcus aureus (MRSA) by bacterial strains, particularly Pseudoduganella sp. NI28 strain for this and multi-drug resistant Staphylococcus aureus (MDRSA) using MIC 5 study. Pseudoduganella sp. NI28 strain, violacein producer, is found in soil mg/L and one of violacein derivative called deoxyviolacein can effect in and has 98% 16S rRNA similarity to Pseudoduganella violaceinigra YIM31327. dyed cotton. The violacein dyed cotton is shown very low antimicrobial The violacein has antibacterial activity against Staphylococcus aureus effects (less than 20%) but deoxyviolacein dyed cotton has 99.9% of MRSA ATCC25923 and other multi drug resistant S.aureus isolated from hospital growth inhibition following KSK0693 methods. Thus deoxyviolacein, violacein patient. Using ethanol extraction, violacein is purified. Antibacterial effect derivative can be used for antibacterial fabrics. 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 C008 Impact of Silicate Solubilizing Burkholderia ebumea CS4-2 Isolation of Lipase Genes from Goat Ruminal Metagenomic on Rice Growth Libraries

Sang-Mo Kang, Raheem Shahzad, Ko-Eun Lee, Yeon-Gyeong Park, Mi-Ra Kwon1, Keun-Sung Kim2, Jin-Sung Lee3, Mi-Rim Park1, Ah-Yeong Kim, Chang-Woo Seo, Sajjad Asaf, and In-Jung Lee* Haesu Ko1, and Kyung-Tai Lee1* School of Applied Biosciences, Kyungpook National University 1Animal Genomics and Bioinformatics Division, National Institute of Animal Science, Rural Development Administration The current study was aimed to isolate and identify the plant root-associated 2Department of Food Science and Technology, Chung-Ang University rhizobacteria and to investigate their potential for silicate solubization, 3Department of Biological Sciences, Kyonggi University growth promotion, IAA Production and encouragement of silicon uptake and deposition inside the plant. Bacterial isolates were selected on the Feed additive is an important pricing decision factor for the animal products basis of their silica solubilizing potential and were identified on micromorphological in the Korean livestock industry. Most feed enzymes, such as phytase, lipase, observation and biochemical characterization. Selected bacterial isolate protease, carbohydrase, depend on import in Korea. Ruminal microbiome was submitted to NCBI and was identified as Burkholderia ebumea CS4-2. is majorly responsible for digestion in ruminants. In particular, ruminal GC-MS results revealed the ability of Burkholderia ebumea CS4-2 to microbiome in goat has been evolved for digestion of wood roughage. To produce high amount of IAA at pH 8. Plant microbe experiment showed a screen and isolate more efficient feed enzymes, metagenomics fosmid significant positive correlation between Burkholderia ebumea CS4-2 and libraries were constructed from ruminal microbiome of three goats. In this silica. Bacterial inoculation with the combination of silica significantly study, we focused on the lipase genes as a kind of feed enzymes. A total of promoted all growth attributes as compared to the water treated plants 57,600 fosmid clones were used for screening lipase activity. These clones and silica fertilized plants. Microscopic observations demonstrated a significant were inoculated on the LB agar plates containing antibiotics of 25 μg/ml difference in silicon on leaf epidermis, indicating that Burkholderia ebumea chloramphenicol and enzymatic substrate of 1% tributyrin using VIAFLO CS4-2 with correlation of silicon resulted in higher Si deposition then silicon 384 and grown at 37°C for 48 h. Circular clear zone around clone indicated fertilized and water treated plants. The current study concluded that the lipid lysis activity. A total of 188 lipase active clones were screened and 39 Burkholderia ebumea CS4-2 has the ability to produce IAA, solubilize clones among them presented strong activity. Strong lipase active fosmid silicate and promote plant growth. This can be the best alternative of clones were sequenced by shotgun pyrosequencing using GS Junior. Sequence synthetic fertilizer as well as in environment clean up. reads were assembled using de novo assembler in Newbler package and [Supported by grants from NRF] 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.

www.msk.or.kr | 27 2016 International Meeting of the Microbiological Society of Korea

C009 C011 Functional Analysis Of Intracellular Nitric Oxide during Effect of Structure and Molecular Weight on the Development in a Filamentous Permeabilizing Ability of Polyethyleneimine (PEI)

Anchalee Pengkit1, Sung-Sil Jeon2, Soo Ji Son3, Soh M. Sandrine and Robert J. Mitchell* 3 1,2 Jae Ho Shin , and Gyungsoon Park * Ulsan National Institute of Science and Technology 1Plasma Bioscience Research Center, Kwangwoon University 2Department of Electrical and Biological Physics, Kwangwoon University Polyethyleneimine (PEI) is used in a wide range of formulations ranging 3Department of Chemistry, Kwangwoon University from washing agents to microbicidal compositions. It is a weakly basic aliphatic polymer known to permeabilize bacterial membranes, property Studies on nitric oxide (NO) are very rare in fungi compared to mammals which makes it useful for a wide range of applications. Some studies reported and plants. In this study, we investigated production of intracellular NO the existence of a difference in degree of permeabilization of bacterial and its possible roles during development of Neurospora crassa, a model membrane when using PEI of different molecular weight and structures. filamentous fungus. Intracellular NO was detected in hypha 8–16 hours We hereby in this study try to evaluate the difference in relative fold after incubation in Vogel’s minimal liquid media and conidiophores during induction possibly observed between PEI of various molecular weights conidiation using a fluorescent indicator (DAF-FM diacetate). NO scavenging and structures. 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 C012 Identification of Protease Genes from Goat Ruminal Characterization of Lactobacillus salivarius Strain Isolated Metagenomic Libraries from Piglet Feces for Probiotic Uses

Mi-Ra Kwon1, Kyung-Tai Lee1, Keun-Sung Kim2, Jin-Sung Lee3, Gwi-Deuk Jin1, Jongbin Park2, and Eun Bae Kim1* 1 1 Mi-Rim Park , and Haesu Ko * 1Department of Animal Life Sciences, Kangwon National University, 2Department 1Animal Genomics and Bioinformatics Division, National Institute of Animal of Animal Life System, College of Animal Life Sciences, Kangwon National Science, Rural Development Administration, 2Department of Food Science and University Technology, Chung-Ang University, 3Department of Biological Sciences, Kyonggi 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 Proteolytic enzymes or proteases are degradative enzymes, catalyzing the growth of pathogenic bacteria when they live in the human/animal guts. hydrolysis of proteins. Protease is the most important industrial enzyme, In this study, we isolated L. salivarius strains from piglets feces in South accounting for approximately 60% of the total enzyme market in the world Korea and characterized their probiotic potentials. We isolated 500 lactic because proteases are widely used in food industries, leather, meat acid bacteria colonies from feces of 10 post-weaning piglets using MRS processing, cheese making, digestive, medical treatment, etc. Also, protease agar plates. Among them, we identified 120 Lactabcillus strains including is one of major feed enzymes in the livestock industry. In this study, we L. salivarius, Lactobacillus plantarum and Lactobacillus reuteri by using screened and identified several proteases from metagenomics fosmid libraries Multiplex-PCR, and 50 L. salivarius strains were selected for further analyses. of goat microbiomes that have been evolved for digestion of wood roughage. Out of the 50 strains, we selected 1 strain, called LS.EBK here, have a high A total of 51,840 fosmid clones were used for screening protease activity. antimicrobial activity against pathogenic Salmonella enterica serovar Typhimurium. Fosmid clones were inoculated on the LB agar plates containing antibiotics LS.EBK strain showed similar growth with other L. salivarius strains. Survival of 25 μg/ml chloramphenicol and enzymatic substrate of 1% skimmilk rates of the strain exposed to MRS broth HCI-adjusted to pH 3.0 (for 30 using VIAFLO 384 and grown at 37°C for 48 h. Circular clear zone around min) and MRS broth containing 0.5% of bile salt (for 30 min) were 0.77% clone indicated skimmilk lysis activity. A total of 29 protease active clones and 0.68%, respectively. These results indicate that L. salivarius LS.EBK were screened and 9 clones among them presented strong activity. Strong strain have a potential characteristics for probiotic uses in livestock industries. protease active fosmid clones were sequenced by shotgun pyrosequencing In the future, we will verify its probiotic effects on piglets by animal trials. using GS Junior. Sequence reads were assembled using de novo assembler [This study was supported by the Strategic Initiative for Microbiomes in in Newbler package and MetaGeneMark web program was used for gene Agriculture and Food, Republic of Korea (914005-04).] annotation. Finally lipase genes were isolated by PFAM and BLASTP Keywords: Probiotic, Lactobacillus salivarius, Salmonella, Antimicrobial search. These protease genes should be developed for industrial application.

28 | www.msk.or.kr Poster

C013 C015 Single-stranded DNA Aptamers Targeting Antimicrobial Deoxyviolacein Mass Production and Aggregation Using Peptides: PG1, PR26 and PMAP36 Fermenter with Escherichia coli Culture

Phat-Loc Nguyen1, Kyeoung-Ah Lee1, Simranjeet Singh Sekhon1, HeeUn Kwon, SeongYeol Choi, and Robert James Mitchell* 2 1 Jiho Min , and Yang-Hoon Kim * School of Biological Science, Ulsan National Institute of Science and Technology 1Department of Microbiology, College of Natural Sciences, 2Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University Violacein is well known antibiotic dye which have effect on antimicrobial, anti-cancer, antiprotozoan and antiviral areas. Deoxyviolacein is one of Aptamers are capable of binding to large molecules such as peptides using violacein derivatives which is produced by various natural violacein producing in detection or chemical modification. In this study, aptamers with 100 bases strain such as J.lividum, C.violaceum, Duganella sp., Pseudoduganella sp., long variable region are employed to target 3 peptides: PG1 (Protegrin-1, and Collimonas sp. It have been discussed that the mass production of an antimicrobial agent in the treatment of local or systemic infections); violacein and its derivative have important value to study. This study is PR26 (Proline-arginine-rich Neutrophil Antibacterial peptide, against enteric focused on the simple mass production of deoxyviolacein with high density gram-negative bacteria) and PMAP36 (Porcine Myeloid Antibacterial peptide, a cell culture of E. coli and the aggregation of deoxyviolacein in cell culture, highly cationic and amphipathic α-helical peptide) which are identified as making it harder to measure exact deoxyviolacein concertration while important components of host defense mechanism [1-3]. After 10 rounds culturing. We made W3110 BH2 strain containing deoxyviolacein-producing of selection, ssDNA aptamers with high affinity were generated by SELEX and bacterial hemoglobin plasmid. This strain presents 0.86 g/L of (Systematic Evolution of Ligands by Exponential enrichment) and were deoxyviolacein production for 20 hours culture in broth. Total deoxyviolacein analyzed using mfold-DNA folding form and surface plasmon resonance amount produced including aggregates is 1.12 g/L in 24 hours. (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 C016 Selection and Characterization of the Glypican-3 Binding Adaptive Laboratory Evolution of Leuconostoc mesenteroides DNA Aptamers J18 in Response to Low Temperature

1 2 2 2 Quang-Thai Nguyen1, Kyeong-Ah Lee1, Sinranjeet Singh Sekhon1, Hye Rim Kim , Ga Yeon Jo , Hey Hee Jeon , and Che Ok Jeon * 1 2 3 Yang-Hoon Kim , Sung-Jin Cho , and Jiho Min * 1Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, 1Department of Microbiology, 2Department of Biology, 3Graduate School of and Center for Food and Bioconvergence, Seoul National University 2 Semiconductor and Chemical Engineering, Chonbuk National University Department of Life Science, Chung-Ang University

Glypicans are heparan sulfate proteoglycans linked to the cell surface and Lactic acid bacteria (LAB) such as Leuconostoc mesenteroides and Leu. play an important role in regulation of cell growth, differentiation, and citreum have been used as kimchi starters to produce kimchi with uniform migration. While six members of the glypican family, Glypican-3 is an efficient quality and good organoleptic properties. However, the use of kimchi diagnostic marker of human hepatocellular carcinomas. Aptamers are starters does not guarantee successful kimchi fermentation because of single-strand oligonucleotides able to bind specifically and sensibility to the competition failure of kimchi starters with indigenous LAB under target molecules, offering great potential for applications in diagnosis and fermentation conditions. Because kimchi is generally fermented at low therapy. In addition, aptamers generally have higher specificity and stability temperatures (0–8°C), an adaptive laboratory evolution approach was for their targets than corresponding antibodies. ssDNA aptamers were used to improve the low temperature adaption. The subcultures of Leu. selected using the Systematic Evolution of Ligands by EXponential enrichment mesenteroides J18 in CDM medium were performed 69 and45 times methodology from an initial library containing a 40 bases long variable representing 300 and 200 generations (total 500 generations) at 8 and region. After in vitro selection, the structural stability as affinity ligands of 6°C, respectively. To characterize the low temperature adaption of strain these aptamers specifically were quantified by strict criteria of equilibrium J18, three substrains with subcultures of 50, 200, and 500 generations (Kd) using surface plasmon resonance and compared with the corresponding were selected and their growths at 6°C were compared with wild type. The monoclonal antibody. genomes of three substrains were sequenced and compared with that of [This study was carried out with the support of "Cooperative Research wild type to identify genes conferring the low temperature adaption. As Program for Agricultural Science & Technology Development (PJ011661)", the results, some unique nucleotide changes were identified from the Rural Development Administration, Republic of Korea.] 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.]

www.msk.or.kr | 29 2016 International Meeting of the Microbiological Society of Korea

C017 C019 Activation of Endophytic-plant Growth-promoting Bacteria Anti-tumor Effect of L-asparaginase Delivered by Salmonella (EPGPB) by Dielectric Barrier Discharge Plasma Treatment typhimurium on Solid Tumors

Sang hye Ji1, Se Chul Chun2, Eun Ha Choi1, and Gyungsoon Park1* Kwangsoo Kim, Daejin Lim, Yeongjin Hong, Kun-Hee Kim, Kyeong il Park, 1Plasma Bioscience Research Center, Kwangwoon University, 2Department of Shinnam Li, Hyun-Ju Kim, Hyon E. Choy, and Jae Ho Jeong* Bioresources and food science, College of Life and Environmental Sciences, Konkuk Department of Microbiology, Chonnam National University Medical School University Bacteria can be engineered to deliver anticancer proteins to tumors via a We investigated the potential of micro Dielectric Barrier Discharge (DBD) controlled expression system that maximizes the concentration of the plasma to increase the effect of isolated endophytic-plant growth-promoting therapeutic agent in the tumor. L-asparaginase (L-ASNase), which converts bacteria (EPGPB) on rice plants. To find the best conditions for the activation asparagine to aspartate and glutamine to glutamate, is an anticancer of EPGPB by plasma treatment, bacteria activity was investigated in protein used to treat acute lymphoblastic leukemia. In this study, Salmonellae accordance with the feeding gases, plasma treatment time and incubation were engineered to express L-ASNase selectively within tumor tissues time. The activity of EPGPB was enhanced more by Air than N2 plasma using the inducible araBAD promoter system of E. coli. Antitumor efficacy treatment and also we were able to confirm the rice seed germination rate of the engineered bacteria was demonstrated in vivo in solid malignancies. and plant growth-promoting activity by Air plasma treated EPGPB. The This result demonstrates the merit of bacteria as cancer drug delivery levels of ROS and RNS from plasma and energy were different depending vehicles to administer cancer-starving proteins such as L-ASNase to be on the kinds of gas supplied to DBD plasma and these might work as effective selectively within the microenvironment of cancer tissue. 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 C020 Inhibition Effect of Lactobacillus plantarum in Colon Cancer Proteobacteria: Diagnostic Marker for Dysbiosis in Gut Microbiota Anna Jeong, Sooyeon Song, and Sejong Oh* Division of Animal Science, Chonnam National University Na-Ri Shin, Tae Woong Whon, and Jin-Woo Bae* Department of Life and Nanopharmaceutical Sciences and Department of Biology, Colorectal cancer is the leading cause of cancer death in Korea because of Kyung Hee University the introduction of Western dietary habits. Particular Lactobacillus strains have beneficial bioactivities in the gastrointestinal tract. The production of Recent advances in sequencing techniques, applied to the study of microbial intracellular reactive oxygen species (ROS) and the amounts of intracellular communities, have provided compelling evidence that the mammalian calcium, protein kinase C activity, cytochrome c, Bid, Bcl-2, Bax, and the intestinal tract harbors a complex microbial community whose composition apoptosis-mediated proteins (caspase-8, caspase-3, and poly ADP ribose is a critical determinant of host health in the context of metabolism and polymerase [PARP]) were evaluated to understand the induction of inflammation. Because an imbalanced gut microbiota often arises from a programmed cell death in HT-29 cells by L. plantarum. The results sustained increase in abundance of the phylum Proteobacteria, the natural obtained from this study indicated that the relative intensities of the human gut flora normally contains only a minor proportion of this phylum. apoptotic-related factors (intracellular ROS and intracellular calcium) and Here, we review studies that explored the association between an abnormal of apoptotic signals (Bax and t-Bid) increased with increasing concentrations expansion of Proteobacteria and a compromised ability to maintain a of the membrane proteins isolated from heat-killed L. plantarum, whereas balanced gut microbial community. We also propose that an increased the relative intensities of cytochrome c, Bcl-2, caspase-8, caspase-3, and prevalence of Proteobacteria is a potential diagnostic signature of dysbiosis PARP decreased. This study determines whether proteins (12 kDa and 15 and risk of disease. kDa) isolated from heat-killed Lactobacillus plantarum induce programmed [This work is supported by a grant from the Mid-career Researcher cell death in HT-29 cells. Proteins isolated from L. plantarum can stimulate Program (2011-0028854) through the National Research Foundation of Korea the apoptotic signals and then consequently induce programmed cell (NRF) and a grant from the Strategic Initiative for Microbiomes in Agriculture death in HT-29 cells. The results in this study suggest that the proteins and Food, Ministry of Agriculture, Food and Rural Affairs, Republic of isolated from L. plantarum could be used as an anti-tumoral agent in Korea.] probiotics and as a component of supplements or health foods.

30 | www.msk.or.kr Poster

C021 C023 Screening and Fermentation Characteristics of Bacillus sp. Combination of Synthetic Genetic Circuitry and Microfluidics with High Amylase and Protease Activity Isolated from for Highly Sensitive Heavy Metal Ion Biosensors Traditional Nuruks Hyun Ju Kim1,2, Ji Won Lim3, Haeyoung Jeong1,4, Sang-Jae Lee5, 6 3,7 1,2 Min Ju Park, Sung Wook Han, Su Jin Heo, Young Ho Hong, Dong-Woo Lee , Taesung Kim , and Sang Jun Lee * Seong Jun Cho, and Seung Won Park* 1Biosystems & Bioengineering Program, University of Science and Technology 2 Life Ingredient & Material Research Institute, CJ Cheil Jedang (UST), Microbiomics and Immunity Research Center, Korea Research Institute Bioscience & Biotechnology (KRIBB), 3Department of Biomedical Engineering, Ulsan National Institute of Science & Technology (UNIST), 4Superbacteria Enzyme producing microorganisms were isolated from many kinds of 5 6 Korean traditional foods. Among the total 1,500 isolated strains, a strain Research Center, KRIBB, Major in Food Biotechnology, Silla University, School of Applied Biosciences, Kyungpook National University, Daegu, Republic of isolated from Nuruks, designated as BA245 was determined to have the 7 most potent ability to present high enzymatic activities for α-amylase and Korea, Department of Mechanical Engineering, UNIST protease. Enzyme activity was examined by using 1% soluble starch and 2% skim milk agar plates. 16S rRNA and gyrase A gene sequence analysis The development of efficient microbial biosensors is essential for proper revealed that strain BA245 belongs to the Bacillus amyloliquefaciens. The integration with micro/nanotechnologies. In order to produce whole-cell optimal activities of its α-amylase and protease were observed at pH 7.0– heavy metal biosensors with improved functions, we must select suitable 9.0 and 37°C. After solid-state fermentation of BA245 on mixed grain for sensory bio-parts and regulatory modules from an enormous quantity of 24 h, the α-amylase and protease activities were 2,762 and 3,868 Units/g. genome information; these components can then be recombined to create The contents of less than 30 kDa peptides of the solid-state fermented synthetic genetic circuits. In this study, multiple copies of a cadC homolog mixed grain increased from 41.95% to 89.66% and 8 kinds of essential free encoding a heavy metal-responsive transcription factor were found in the amino acids were increased 13 times compared to the raw materials. genome of Bacillus oceanisediminis 2691, and the heavy metal specificities of [This study suggested that solid-state fermentation using strain BA245 can the encoded proteins were characterized using a fluorescence reporter assay. be used for industrial applications due to their activity in production of For intracellular signal amplification, we constructed CadC- controlled T7 grain enzyme-containing foods.] 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].

C022 C024 Effect of Metabolic Condition Medication and Probiotics Lactobacillus plantarum Suppressed ß-hexoaminidase, Formulation on the Model Rats and Their Intestinal Microbiota Histamine, and Expression of TNF-α and IL-4 in

Seokcheon Song1, Joohyun Shin2, Jaegu Seo2, BPA-stimulated RBL-2H3 Cells Myungjoon Jung2, and Eungbin Kim1* Jin A Lim and Sejong Oh* 1Department of Systems Biology, Yonsei University, 2R&D Center, Cellbiotech, Co. Ltd. Division of Animal Science, Chonnam National University of Korea

Nowadays metabolic condition medications were consumed by patients This study inspected the inhibitory effect of the glycoprotein isolated from in developed countries and some have side effect on intestine. It is known Lactobacillus plantarum on bisphenol A tempted allergic inflammatory gut microbiota is important to intestinal health and related with diseases. reaction in RBL-2H3 cells. RBL-2H3 cells were treated with Bisphenol A (50 Commercially available probiotics, Duolac-Gold (DG) was used to examine μmol/ml) and the glycoprotein isolated from L. plantarum (5–100 µg/ml) effects of the drugs since host diet can affect on shaping gut ecology. Two for 30 min. histamine release, β-hexosaminidase and intracellular Ca2+ drug ingredients, leflunomide (LF), amlodipine (AMD), was chosen based on side level were estimated in the medium. Activities of PKC, iNOS were measured effect profile, preliminary experiment. Here, we conducted in vivo experiment by western blotting. Also IL-4 and TNF-α were measured by RTq-PCR. In about effects of LF, AMD, DG on gut, gut microbiota with a rat model. this study, glycoprotein isolated from L. plantarum inhibited the histamine Rats were distributed in 7 groups: control, LF-administered, LF plus low, release, β-hexosaminidase, the intracellular Ca2+ level, and activity of high dose of DG-administered, AMD-administered, AMD plus low, high iNOS in bisphenol-A treated RBL-2H3 cells. The results indicated that the dose of DG-administered. glycoprotein isolated from L. plantarum inhibits expression of cytokines Target probiotic, gram positive, negative microbes in stool were quantified. associated with allergy such as TNFα and IL-4 on the degranulation of mast The probiotics decreased by LF, AMD but DG offset the effect of the drugs. cell. In conclusion, the results showed that degranulation, histamine release LF, AMD decreased gram negative microbes but they were recovered by were inhibited by the glycoprotein of L. plantarum in the BPA stimulated DG. In metabolic data, decrease of weight of rat by AMD was observed. RBL-2H3 cells, while amount of Ca2+ and iNOS were diminished. TNFα and Jejunum was inflated by AMD and it is a good agreement with reduced IL-4, two of allergy-related cytokines were suppressed. Taken together weight. Ileal ulcer by LF and AMD was also observed. All changes above by might help to prevent allergy diseases induced by environmental hormone medications were alleviated by DG. To examin ulcer closely, cytokines in such as BPA. 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]

www.msk.or.kr | 31 2016 International Meeting of the Microbiological Society of Korea

C025 C027 Total Protein Isolated from Lactobacillus plantarum Has a Determination of Antiviral Effects of Adenosine Analogues Protective Character from Cadmium Chloride-stimulated on Epstein-Barr Virus Infection Raw 264.7 Cells Miyeon Cho, Seok-Won Jung, Soomin Lee, and Hyojeung Kang* Miyoung Shin and Sejong Oh* College of Pharmacy and Institute of Microorganisms, Kyungpook National Division of Animal Science, Chonnam National University University

The food and water we consume are often contaminated with a range of Cordyceps species are known to produce numerous active components chemicals and heavy metals, such as lead, cadmium, arsenic, chromium, and are used for diverse medicinal purposes because of their varied and mercury that are associated with numerous diseases. Especially, physiological activities, including their anticancer, anti-inflammatory, and cadmium is well known as one of the major components can cause a antimicrobial effects. Cordycepin, one of adenosine analogues, differs number of lesions in many organs. However, the mechanism of cadmium-toxicity from adenosine in that its ribose lacks an oxygen atom at the 3' position. and its inhibition are not clear. In this study, we demonstrated that total Cordycepin has been reported to be a main effector molecule in Cordyceps protein isolated from lactobacillus plantarum protects raw 264.7 cells extracts that executes antiviral activities against several viruses including from expression of inflammation-related factors stimulated by cadmium influenza virus, plant viruses, human immunodeficiency virus (HIV), murine chloride (100 µM). Furthermore, we evaluated the cytotoxicity of cadmium leukemia virus, and Epstein-Barr virus (EBV). In addition, adenosine and its using MTT assay and intracellular Ca2+ using fluorescence, and activities of deaminase inhibitor showed strong antiviral potentials that were about activator protein PKC-α, iNOS, (AP)-1, MAPK, PCNA, and cyclin D1/CDK4 4,000 times more potent than the activity of the direct inhibitory effect by using immunoblot. The results obtained from this experiment indicated adenine arabinoside on Herpes simplex virus (HSV)-1, suggesting adenosine that total protein isolated from L. plantarum inhibits the intracellular Ca2+ itself plays an important role to produce antiviral activities. In this study, mobilization. Also, it significantly suppressed inflammatory factors [expression we show that cordycepin and adenosine possess antiviral and antitumor of AP-1 (c-Jun and c-Fos), MAPKs (ERK, JNK and p38), iNOS]. With regard to activities against EBV and EBV-associated gastric carcinoma, respectively. cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly Furthermore, we report epigenetic mechanisms used by cordycepin and suppressed by treatment with total protein isolated from L. plantarum in adenosine to exert their antiviral and antitumor effects. the presence of Cd. Taken together, the findings suggested that total [This study was supported by grants from KRF (2015R1D1A3A01020679).] 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.

32 | www.msk.or.kr Poster

D001 D003 Effect of Sub Minimal Inhibitory Concentration Ferroportin Promote ROS Influx into Salmonella Containing Chlorhexidine on Binding Characteristics of Streptococci and Vesicle Resulting in Intramacrophage Bacterial Killing Actinomycetes Daejin Lim, Hyun-Ju Kim, Jea-Ho Jeong, Kwangsoo Kim, Kun-Hee Kim, So Yeon Lee and Si Young Lee* Kyeongil Park, Shinan Li, and Hyon E. Choy* Department of Oral Microbiology, Colleage of Dentistry, Gangneung-Wonju Department of Microbiology, Chonnam National University Medical School National University In response to microbial infection, defensin-like peptide hepcidin is induced Chlorhexidine has been used for a long time as mouth washes for the to limit the serum ironby reducing iron release from macrophages through control of dental caries, gingivitis and dental plaque. Minimal inhibitory interleuklin-6 (IL-6) signaling.Here, we reportthat a nuclear receptor ERRγ concentration (MIC) is the lowest concentration of antimicrobial substance mediates Salmonella infection through host iron alteration by hepcidininduction, to inhibit the growth of bacteria. The concentrations lower than the MIC and demonstrate an antimicrobial effect of ERRγ inverse agonist. Hepatic are called as sub minimal inhibitory concentrations (sub-MICs). Many ERRγ geneexpression was induced by Salmonella-stimulated IL-6 signaling, studies have reported that sub-MICs of antimicrobial substances can inhibit resulting in an induction of hepcidin, and degradation of ferroportin on virulence factors of bacteria. The aim of this study was to investigate the macrophage cell membrane, and eventual hypoferremia in mice. An effect sub-MIC of chlorhexidine on biofilm formation by each bacterial inverse agonist of ERRγ restored the Salmonella-mediatedhypoferremia strains and coaggregation of Streptococcus gordonii, Streptococcus mutans, through reduction of ERRγ-mediated hepcidin gene expression, and exerted a Actinomyces naeslundii and Actinomyces odontolyticus. The biofilm formation potentantimicrobial effect on the Salmonella infection, thereby improving of S. gordonii, A. naeslundii and A. odontolyticus was not affected by host survival. Further study indicated that mechanism underlying antimicrobial sub-MIC chlorhexidine. However, the biofilm formation of S. mutans was effect of inverse agonist of ERRγ was to induce ROS production in Salmonella increased after incubation with sub-MIC chlorhexidine. Coaggregation of Containing Vesicle by modulating iron content therein, consequence of A. naeslundii with A. odontolyticus has been reduced by sub-MIC chlorhexidine. which was enhanced bacteria killing. Coaggreagation of A. naeslundii with S. gordonii had not affected. These [This work was supported by NRF-2014R1A2A1A10051664.] results indicate that sub-MIC chlorhexidine could influence on the binding properties such as biofilm formation and coaggregation in Streptococci and Actinomyces.

D002 D004 Whole Genome-scale Transcriptomic Analysis of c-di-GMP Inhibition of Varicella-zoster Virus Replication by the Extract Signaling in Enteropathogenic Escherichia coli from Elaeocarpus sylvestris

Hyung Tae Lee, Dalmuri Han, June Bong Lee, Na-Eun Kim, So-Hee Bae, June-Eun Kim, and Yoon-Jae Song* Chong-Hae Hong, and Jang Won Yoon* Department of Life Science, Gachon University College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University Varicella-Zoster virus (VZV), which is a member of the α-herpesvirus subfamily, causes varicella (chickenpox) by primary infection and reactivates Enteropathgenic Escherichia coli (EPEC) is in the family of Enterobacteriaceae to cause herpes-zoster (shingles). To identify a new anti-VZV drug candidate that often cause diarrheal diseases of young children in developing countries. or develop a substitute for existing medicines, seventy percent ethanol The main routes of EPEC transmission are consumption of the contaminated extracts from plant materials were screened for their inhibitory activities food, water, or fomites as well as direct contact with infected animals. on VZV replication in vitro. Crude ethanol, EtOAc fraction and hot water EPEC can sense and respond to various environmental cues by signaling extracts of Elaeocarpus sylvestris leaves (ESE) distinctly inhibited the replication through the small molecules. Cyclic diguanylate (c-di-GMP) is one of such of the VZV pOka strain without showing any significant cytotoxic effect signaling molecules present in bacteria, but absent in eukaryotes or against MRC-5 cells. Furthermore, ESE considerably down-regulated VZV archaea, which modulates several cellular functions. However, the role of immediate early (IE) gene expression. Thus, ESE has a promising antiviral c-di-GMP signaling in the pathophysiology of EPEC has not been fully activity against VZV by reducing VZV IE gene expression and replication. understood. Here we investigated the role of c-di-GMP signaling in the [This research was supported by Bio-industry Technology Development EPEC prototype strain, E2348/69, using genome-wide transcriptomics by Program, Ministry of Agriculture, Food and Rural Affairs (No. 311063-5).] 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.]

www.msk.or.kr | 33 2016 International Meeting of the Microbiological Society of Korea

D005 D007 The Quorum Sensing-dependent Factor(s) Can Modulate Eukaryotic Stress Response Gene ATF3 Provides Protection the Activity of Protease IV, a Major Virulence Factor of from Staphylococcus aureus and Listeria monocytogenes Pseudomonas aeruginosa Infections

Jungmin Oh1,2,3, Soo-Kyung Kim1,2,3, Xi-Hui Li1,2,3, and Joon-Hee Lee1,2,3* Sung-Yoep Lee, Suhkneung Pyo, and Dong Kwon Rhee* 1Lab of Microbiology, 2College of Pharmacy, 3Department of Pharmacy, Pusan School of Pharmacy, Sungkyunkwan University National University Activating transcription factor (ATF)-3 is a stress-induced transcriptional Pseudomonas aeruginosa can infect plants and animals, including humans. regulator. The role of ATF3 in cancer has been well defined, but how ATF3 In humans, it can cause several disease on various body organ, such as the functions in bacterial infection is not well understood. Pneumococcal corneas the lungs and the kidneys. The major factor of the Pseudomonas infection has been shown to induce ATF3 expression, which subsequently corneal infection is the Protease IV (PIV). PIV is a quorum sensing (QS)-regulated enhances cytokine production and provides protection from lethal Streptococcus exoprotease and a key virulence factor against Tenebrio molitor, an insect. pneumoniae infection, but the role of ATF3 in other Gram-positive (G+) PIV expressed in QS mutant (MW1, lasI‾,rhlI‾ double mutant of P. aeruginosa) infections remains unclear. Here, we report that infection with other G+ showed much reduced activity. The cell-free culture supernatant (CFCS) bacteria (Staphylococcus aureus and Listeria monocytogenes) and with G- from the PIV-overexpressing WT strongly induced the melanization of T. bacteria (Escherichia coli as a negative control) also significantly induced molitor larva, but the CFCS from the PIV-overexpressing MW1 rarely ATF3 expression. Moreover, the production of cytokines (tumor necrosis induced the melanization, even though it contained comparable amount factor alpha [TNF]-α, interleukin [IL]-1β, IL-6, and interferon [IFN]-γ) was of PIV to CFCS of WT. PIV is the lysyl endopeptidase, so we compared the enhanced by ATF3 in S. aureus and L. monocytogenes infection, but decreased activities of PIV-overexpressing in WT and MW1 using chromogenic in E. coli infection. In addition, in S. aureus and L. monocytogenes substrate {N-(p-Tosyl)-Gly-Pro-Lys 4-nitroanilide acetate salt}. PIV-overexpressing infections, ATF3 WT mice cleared bacteria more efficiently and had higher WT showed strong PIV activity, but PIV-overexpressing CFCS in MW1 was survival rates than ATF3 knockout (KO) mice. However, in E. coli infection, not. When we purified PIVs from WT and MW1, the activity of PIV purified no significant difference was found in survival rate. Taken together, these from MW1 was significantly lower than PIV from WT. Interestingly, when data suggest that ATF3 provides protection from S. aureus and L. monocytogenes we add CFCS of piv mutant to the PIV purified from MW1, the activity of infections; however, the role of ATF3 in E. coli infection is more complicated PIV from MW1 was restored to the level of the PIV from WT. We therefore and should be further elucidated. suggest that there are some QS-dependent factors that modulates the [Supported by NRF-2015R1 A2 A1 A10052511.] Protease IV activity. [Supported by grants from KRF]

D006 D008 A Salmonella Virulence Protein Interacts with PhoR That Isolation of New Bacteriophages to Control Pathogenic Activates Pho-regulon Bacteria, Bacillus cereus

Soomin Choi Jeong-A Lim, Sojung Kim, Jonguk Kim, Jisoo Hong, Eunjung Roh, Microbial Genetic Laboratory, College of Life Science, Kyung Hee University Kyu Seok Jung, Jae-Gee Ryu, and Jin-Woo Park* Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Several intracellular pathogens, including Salmonella enterica require the Administration virulence protein MgtC to survive within macrophages and to cause a lethal infection in mice. We now report that MgtC activates Pho-regulon Bacillus cereus is pathogenic bacteria that causes diarrhea and vomiting including PhoB-PhoR two component system that expressed Phosphate and contaminates various vegetables in addition to foods. Generally, regulator gene. PhoR is a histidine kinase sensor protein that appears to sprouts were cultivated in hydroponics because of its advantages like even respond to periplasmic orthophosphate. PhoR respond phosphate from transmission of nutrient to plant. However, if some pollutants like bacteria periplasm, auto-phosphorylate to histidine, and phosphorylate response were contaminated to media all of the sprouts in the same system also regulator protein PhoB, then phosphate regulator gene has been activated. contaminated. In deed in our study, media contamination was connected We establish that MgtC interacts with the a subunit of the Pho-regulon, to sprout contamination. After the seeds of Chinese cabbage were PhoR, activated phosphorylation of gene regulator protein, PhoB. 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.]

34 | www.msk.or.kr Poster

D009 D011 The Putative Fungicidal Molecules Show an Antifungal Effect Ohmyungsamycins, New Antimycobacterial Cyclic Peptides, on Candida albicans Virulence Through a Regulating the Activate Autophagy via AMP-activated Protein Mitochondrial Activity Kinase-mediated Signaling

Young Kwang Park1, Se Woong Kim1, Hwang Suk Kim2, Tae Sung Kim1,2, Yern-Hyerk Shin3, Hye-Mi Lee1,2, Soohyun Um3, Hee-Yoon Lee2, and Joon Kim1* Jin Kyung Kim1,2, Dong-Chan Oh3, and Eun-Kyeong Jo1,2* 1Lab. of Biochemistry, Division of Life Sciences, Korea University, 2Department of 1Department of Microbiology, College of Medicine, Chungnam National University, Chemistry, KAIST 2Infection Signaling Network Research Center, 3Natural Products Research Institute, College of Pharmacy, Seoul National University The opportunistic pathogen Candida albicans colonizes the human skin and human mucosal surface. Undergoing morphogenesis from yeast to The AMP-activated protein kinase (AMPK) pathway activation is required hyphal form in various environmental conditions is one of the well-known for antimicrobial host defense against Mycobacterium tuberculosis (Mtb), virulence factors of C. albicans. Small molecules were designed as chemical a major pathogenic strain of human tuberculosis. During search for novel structural analogues of Bafilomycin which is a V-ATPase specific inhibitor. AMPK-activating agents with antimycobacterial effects, we had an attention To characterize small molecules, we investigated the effect of small on the newly identified Ohmyungsamycins (OMS) A and B, new cyclic molecules on growth rate and the morphological transition. Growth and peptides with antibacterial and anticancer effects. In this study, we show hyphal formation rate were decreased by treatment of small molecules. that OMS are essential autophagy activator leading to antimicrobial responses Moreover in the candidiasis murine model, molecule C has shown the against Mtb, through activation of AMPK pathway in murine bone marrow- effective antifungal activity. The microarray result which was using a set of derived macrophages (BMDMs). OMS robustly activated autophagy induction RNA from control and drug-treated cells revealed mitochondrial related and upregulated colocalization of LC3 autophagosomes with bacterial genes are influenced by molecule C. Also in 2D gel analysis data has shown phagosomes in BMDMs. The activation of autophagy was required for the similar results as microarray data. As a conclusion from these results, OMS-induced antimicrobial responses against Mtb in BMDMs. We further molecule C has the antifungal effect and it appears to influence on showed that OMS triggered AMPK activation, which was essential for mitochondrial activity. Further investigation on the mechanism will be OMS-mediated autophagy activation. Collectively, these data show that presented in this study. OMS is a promising candidate for new antimycobacterial therapeutics through autophagy activation via AMPK dependent signaling.

D010 D012 Vacuoles are Essential for Morphogenesis and Virulence in Antagonistics against Pathogenic Fusarium solani and Candida albicans Fusarium oxysporum by Novel Peptides from Bacillus amyloliquefaciens PT14 Se Woong Kim, Young Kwang Park, Yoo Jin Joo, Yu Jin Chun, Ju Yeon Hwang, Je-Hyun Baek, and Joon Kim* Hee Kyoung Kang1 and Yoonkyung Park1,2* Lab of Biochemistry, Division of Life Sciences, Korea University 1Department of Biomedical Science, Chosun University, 2Research Center for Proteineous Materials, Chosun University The human opportunistic fungal pathogen Candida albicans undergoes a morphological transition under various conditions. Moreover, it is well Bacillus species have recently drawn attention due to their potential use in known that morphogenesis is important for the onset of pathogenesis of the biological control of fungal diseases. This paper reports on the antifungal C. albicans. In this study it is shown that filamentous growth critical for activity of novel peptides isolated from Bacillus amyloliquefaciens PT14. pathogenesis of C. albicans requires active intrinsic vacuole function. Reverse-phase high-performance liquid chromatography revealed that B. Using biochemical analyses, it was determined that the VMA4 and VMA10, + amyloliquefaciens PT14 produces five peptides (PT14-1, -2, -3, -4a, and putative E and G subunits of the vacuolar H -ATPase complex, genes were -4b) that exhibit antifungal activity but are inactive against bacterial overexpressed in a hyphal growing cell compared to that of a yeast strains. In particular, PT14-3 and PT14-4a showed broad-spectrum antifungal growing cell. Deleting VMA4 or VMA10, both of which form a stalk domain activity against Fusarium solani and Fusarium oxysporum. The PT14-4a in the complex, abolished intrinsic vacuolar functions, such as endosomal N-terminal amino acid sequence was identified through Edman degradation, acidification and trafficking. Moreover, Vma4 and Vma10 were important and a BLAST homology analysis showed it not to be identical to any other for morphological conversion and hyphal-specific gene expression. These protein or peptide. PT14-4a displayed strong fungicidal activity with deletion mutants were also characterized as avirulent in mouse experiments. minimal inhibitory concentrations of 3.12 mg/L (F. solani) and 6.25 mg/L Furthermore, VMA4 and VMA10 deletion caused hypersensitive growth in (F. oxysporum), inducing severe morphological deformation in the conidia the presence of fluconazole, suggesting that they are involved in the and hyphae. On the other hand, PT14-4a had no detectable hemolytic resistance to antifungal drugs. Based on these findings, roles were found activity. This suggests PT14-4a has the potential to serve as an antifungal for Vma4 and Vma10 of C. albicans in vacuole biogenesis, vacuole-dependent agent in clinical therapeutic and crop-protection applications. 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.

www.msk.or.kr | 35 2016 International Meeting of the Microbiological Society of Korea

D013 D015 The Impact of Serum Albumin on Predation by Bdellovibrio Genome-wide Transcriptome Analysis of Sch9-dependent bacteriovorus HD100 Thermotolerance Mechanism Reveals the Dual Functional Heat Shock Factor 1, Hsf1, in Cryptococcus neoformans Ga Young Cho, Hansol Im, Ajay Monnappa, and Robert Mitchell* Dong-Hoon Yang1, Kwang-Woo Jung1, Soohyun Bang1, Jang-Won Lee1, Ulsan National Institute of Science and Technology Min-Hee Song1, Yeonseon Lee1, Eunji Jeong1, Anna Floyd2, Richard Festa3, Giuseppe Ianiri4, Alex Idnurm4, Dennis Thiele3, Joseph Heitman2, and Bdellovibrio bacteriovorus HD100 is predatory bacterium and is considered Yong-Sun Bahn1* as a potential living antibiotic. As many bacteria can cause an infection 1Department of Biotechnology, Center for Fungal Pathogenesis, Yonsei University, within the blood, its components are important factors that may influence 2Departments of Molecular Genetics and Microbiology, Medicine, and Pharmacology the predation activity. We show here, based upon predation patterns and and Cancer Biology, Duke University Medical Center, Durham 27710, NC, USA, 3 microscopic analyses, that serum albumin binds to B. bacteriovorus HD100 Department of Pharmacology & Cancer Biology and Biochemistry, Medicine, and Phamacology and Cancer Biology, Duke University Medical Center, Durham and severely impacts the predation of E. coli. 4 27710, NC, USA , Division 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).]

D014 D016 Transcriptomic Profiles from Bdellovibrio bacteriovorus Unravelling of the Target of Rapamycin (TOR1) Kinase Signaling HD100 during Predation on an Extended-Spectrum Pathway in Human Fungal Pathogen Cryptococcus neoformans Beta-Lactamase (ESBL) Strain of Escherichia coli Yee-Seul So1, Giuseppe Ianiri2, Alex Idnurm2, Jae-Hyung Jin3, and Yong-Sun Bahn3* 1 1 2 1 Department of Biotechnology, College of Life Science and Biotechnology, Yonsei SooIn Choi , Mohammed Dwidar , and Robert J. Mitchell * 2 1 University, Division of Cell Biology and Biophysics, School of Biological School of Life Science, Ulsan National Institute of Science and Technology 3 Sciences, University of Missouri-Kansas City, MO 64110, USA, Department of 2Okinawa Institute of Science and Technology, Japan Biotechnology, Center for Fungal Pathogenesis, Yonsei University

Bdellovibrio bacteriovorus is a Gram-negative bacterium considered to be The TOR pathway has been implicated in regulating cellular responses to a potential living antibiotic because of their unique predatory life and nutrients, including proliferation, translation, autophagy, and ribosome ability to attack a wide range of Gram-negative bacteria. In this regard, biogenesis. Here we identified two homologues of S. cerevisiae Tor, CNAG_ predatory bacteria are being considered as an alternative to conventional 06642 (Tor1) and CNAG_05220 (Tlk1, TOR-like kinase 1), in Cryptococcus antibiotics against multi-drug resistant pathogens. In this study, we neoformans. To study the TOR-1 signaling pathway, we attempt to evaluated the predation mechanisms used by B. bacteriovorus HD100 construct the tor1Δ and tlk1Δ mutants and phenotypically analyze them. while predating on an extended-spectrum beta-lactamases (ESBL) producing Although we fail to construct the tor1Δ mutant, we successfully construct Escherichia coli via whole RNA-sequencing. the tlk1Δ mutant. The essentiality of TOR1 is independently confirmed by [Supported by grants from DARPA] 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]

36 | www.msk.or.kr Poster

D017 D019 Kinome Webs Reveal Novel Pathogenicity Networks in Production and Characterization of Sodium Hydroxide Human Fungal Pathogen Cryptococcus neoformans Induced Vibrio parahaemolyticus Ghosts as a Potential Kyung-Tae Lee1, Yee-Seul So2, Dong-Hoon Yang2, Kwang-Woo Jung2, Vaccine Candidate Jaeyoung Choi3, Dong-Gi Lee4, Hyojeong Kwon2, Juyeong Jang2, Li Li Wang2, Soohyun Cha2, Gena Lee Meyers2, Joohyeon Hong2, Hyun Jung Park, Seongmi Ji, Nagarajan Vinod, Sung Oh, Jung Mo Koo, Soohyun Bang2, Je-Hyun Ji2, Goun Park2, Hyo-Jeong Byun2, Sung Woo Park2, Han Byul Noh, Ki-Sung Lee, Sei Chang Kim, and Chang Won Choi* Young-Min Park2, Gloria Adedoyin5, Taeyup Kim5, Anna K Averette5, Department of Biology & Medicinal Science, Pai Chai University Jong-Soon Choi4, Eunji Cheong2, Yong-Hwan Lee3, and Yong-Sun Bahn2* 1 2 Vibrio parahaemolyticus PCU-1 ghosts (VPGs) were generated by chemically- Department of Biotechnology, Center for Fungal Pathogenesis, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, induced lysis and the method is based on minimum inhibitiory concentration 3Department of Agricultural Biotechnology, Seoul National University, 4Biological (MIC) of sodium hydroxide (NaOH), acetic acid (CH3COOH), boric acid Disaster Analysis Group, Korea Basic Science Institute, 5Department of Molecular (BH3O3), citric acid (C6H8O7), maleic acid (C4H4O4), hydrochloric acid (HCl) Genetics and Microbiology, Medicine, and Pharmacology and Cancer Biology, Duke and sulphuric acid (H2SO4). The MICs of NaOH, CH3COOH, BH3O3, C6H8O7, University Medical Center, USA 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 Cryptococcus neoformans is the leading cause of death by fungal of V. parahaemolyticus cells was reached 100% at 10 min after treatment meningoencephalitis; however, treatment options remain limited. To of other chemicals. Nevertheless, real-time PCR confirmed that NaOH understand its underlying pathogenicity mechanisms and identify novel only induced 100% DNA-free VPGs. Therefore, NaOH was selected as the therapeutic targets, we have constructed 226 signature-tagged gene-deletion best chemical to produce VPGs as a potential vaccine candidate. The strains for 114 putative kinases and examined their phenotypic traits under 30 formation of the trans-membrane lysis tunnel structure in NaOH induced distinct in vitro growth conditions, in two different host environments. Phenotypic VPGs, which was observed by scanning electron microscopy. SDS-PAGE clustering analysis not only identified several novel kinases in known and agarose gel electrophoresis supported that cytoplasmic proteins and signalling pathways but also hitherto uncharacterized signalling cascades. genomic DNA released from NaOH induced VPGs to culture medium through Our large-scale virulence assays using an insect model and signature-tagged the tunnel structure. In conclusion, the results of this study suggest that mutagenesis-based infectivity assays in a murine model discovered 60 the NaOH induced VPGs are faster, more economical and safer method pathogenicity-regulating kinases in diverse biological categories: growth and the than protein E-mediated lysis cassette in inactivated vaccine research. cell cycle, nutrient metabolism, the stress response and adaptation, cell [This work was supported by the Human Resource Training Program for signalling, cell polarity and morphology, vacuole trafficking, tRNA modification, Regional Innovation and Creativity through the Ministry of Education and and other previously unknown functions. Our comprehensive fungal kinome analysis National Reserch Foundation of Korea (2015035949).] provides insights into the pathobiological signalling circuitry of C. neoformans and suggests novel anticryptococcal or antifungal drug targets. D018 D020 Detection and Growth Inhibition of Streptococcus mutans Production and Characterization of Hydrochloric Acid by Aptamers Induced Listeria monocytogenes Ghosts (LMGs) as a Potential Vaccine Candidate kyeong-Ah Lee1, Simranjeet Singh Sekhon1, Gna Ahn1, Ji-Young Ahn1, 1 2 3 Yang-Hoon Kim , Sung-Jin Cho , and Jiho Min * Seongmi Ji, Hyun Jung Park, Nagarajan Vinod, Sung Oh, Jung Mo Koo, 1Department of Microbiology, 2Department of Biology, 3Graduate School of Han Byul Noh, Ki-Sung Lee, Sei Chang Kim, and Chang Won Choi* Semiconductor and Chemical Engineering, Chonbuk National University Department of Biology & Medicinal Science, Pai Chai University

The Streptococcus mutans bacterial species plays a leading role in the Listeria monocytogenes ghosts (LMGs) were generated by chemically initiation of dental caries. Among important virulence factors of this induced lysis and the method is based on minimum inhibitiory concentration pathogen, its ability to form the biofilm is vital not only to its survival and (MIC) of sodium hydroxide (NaOH), acetic acid (CH3COOH), boric acid (BH3O3), persistence in the oral cavity, but also for its pathogenicity as well. Here citric acid (C6H8O7), maleic acid (C4H4O4), hydrochloric acid (HCl) and we developed multifunctional aptamer for growth inhibition and detection sulphuric acid (H2SO4). The MICs of NaOH, CH3COOH, BH3O3, C6H8O7, of Streptococcus mutans. Aptamers are single-stranded oligonucleotides C4H4O4, HCl, and H2SO4 were 6.25, 6, 25, < 50, 25, 12.5, 6.25, and 12.5 mg/ml, that interact with target molecules with high affinity and specificity. With respectively. Among those, the lysis efficiency of L. monocytogenes cells advantages in targeted therapy and diagnosis, aptamers are considered as was reached 100% at 5 min after treatment of H2SO4, 10 min after treatment great potential ligand. We collected DNA aptamer that bind to S. mutans of NaOH, 15 min after treatment of HCl and 30 min after treatment of and developed the aptamer-based detection assay for specific detection maleic acid. The formation of the trans-membrane lysis tunnel structure in of the cell. Moreover, several of aptamers inhibit cell growth of S. mutans respective chemicals induced LMGs, which was observed by scanning electron more than control. This assay with high specificity can be used as an microscopy. SDS-PAGE and agarose gel electrophoresis supported that alternative method for the therapy and detection of oral disease. cytoplasmic proteins and genomic DNA released from H2SO4, NaOH and [This work was carried out with the support of "Cooperative Research HCl induced LMGs, respectively, to culture medium through the tunnel Program for Agriculture Science & Technology Development (Project title: structure. Nevertheless, real-time PCR confirmed that HCl only induced Risk Assessment Research and Development of Rapid Diagnostic Method 100% DNA-free VPGs. Therefore, HCl was selected as the best chemical to for Biological, Chemical and Environmental Animal Disease, Project No: produce VPGs as a potential vaccine candidate. PJ01052301) Rural Development Administration, Republic of Korea. This [This work was supported by the Human Resource Training Program for work was supported by the Human Resource Training Program for Regional Regional Innovation and Creativity through the Ministry of Education and Innovation and Creativity through the Ministry of Education and National National Reserch Foundation of Korea (2015035949).] Research Foundation of Korea (NRF-2015H1C1A1035921).]

www.msk.or.kr | 37 2016 International Meeting of the Microbiological Society of Korea

D021 D023 Identification and Mechanism of LL37 and Its Analogs with Effect of HAMP and T1AMP on the Antimicrobial Activity Potent Antimicrobial Activity against Acinetobacter against Pseudomonas aeruginosa and Multidrug-resistant baumannii Strains Pseudomonas aeruginosa

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

The emergence of multidrug-resistant Acinetobacter baumannii is recently Antimicrobial peptides (AMPs) are important molecules of the host defense becoming increasingly important as nosocomial infection and has affected system against invading pathogen. Antimicrobial peptides are small molecules people with compromised immune system. Thus, effective novel antimicrobial containing amino acids 10 to 50 amino acid residues with positive charge agents are urgently required. Cationic antimicrobial peptides (CAMPs) are (generally +2 to +10), which exhibit antimicrobial activity against Gram- thought to play an important self- defense role in many organisms. Because negative bacteria, Gram positive bacteria, yeasts and fungi and viruses. In of their ability to disrupt the bacteria membrane, leading to cytoplasmic this study, antimicrobial peptide HAMP was identified from the scorpion disruption and cell death, so AMPs provide an alternative to existing antibiotics. Heterometrus petersii, which is an amphipathic α-helical structure and However, instability by proteases generated by human inherently restrict antimicrobial activity. Base on this parent peptide HAMP, we designed the AMPs. We therefore designed novel cationic antimicrobial peptides by analogs peptide T1AMP by truncation and substitution. The analog peptide substituting residues in truncated human cathelicidin LL37. The antimicrobial T1AMP have strong antimicrobial activity against Gram negative bacteria activities, anti-biofilm activities of the five antimicrobial peptides were including Pseudomonas aeruginosa and multidrug-resistant Pseudomonas tested against Acinetobacter baumannii. Moreover, these peptides were aeruginosa. Furthermore, these peptide a little hemolytic activity on red found to be resistant to proteolytic degradation, but did not completely blood cell and low cytotoxicity of HaCaT cell. Taken together, the results of exhibited hemolytic activity and were not cytotoxic to normal human this study indicate that the HAMP and analog peptide T1AMP may have keratinocytes except for LL37. LL37 and its analogs appeared α-helical or β- antimicrobial activity and effective therapeutic agent against multidrug- sheet structures within bacterial membranes by Circular dichroism analysis. resistant Pseudomonas aeruginosa. 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 D024 Characterization of Antimicrobial Activity and Mechanism Antimicrobial Activity, Anti-biofilm and Action Mechanism of Antimicrobial Peptide against Bacteria of Charge-enriched AMPs against Both Gram-positive and Gram-negative Bacteria with Therapeutic Potential for 1,2 1,2 Su Jin Ko and Yoonkyung Park * Clinical Antibiotic-resistant 1Research Center for Proteineous Materials (RCPM), 2Department of Biotechnology and BK21-Plus Research Team for Bioactive Control Technology, Chosun Hyo Mi Han1 and Yoonkyung Park1,2* University 1Research Center for Proteineous Materials (RCPM), 2Department of Biotechnology Bacterial drug resistance is a major problem in human health as it has led and BK21-Plus Research Team for Bioactive Control Technology, Chosun University to the reduced efficacy of conventional antibiotics. Thus, the identification of novel antibiotics is essential. Antimicrobial peptides are found in various Cationic antimicrobial peptides (AMPs) are essential components of the living organisms, including plant, insects, amphibians, and mammals, innate immune system, offering protection against invading pathogenic where the play important roles in defense. Antimicrobial peptide (AMP) bacteria. In nature, AMPs serve as antibiotics with broad-spectrum antimicrobial have been increasing interest as alternative to conventional antibiotics and anti-biofilm properties. However, low effective stability in high-salt due to their broad spectrum antimicrobial activity and reduced possibility environments and physiological instability in biological membranes limit for the development of bacterial drug resistance. A novel antimicrobial the applicability of naturally occurring AMPs as novel therapeutics. We peptide named MAC was isolated from the venom of the solitary bee therefore designed short synthetic cationic peptides by substituting key Macropis fulvipes. MAC exhibited antimicrobial activity against both residues in myxinidin, an AMP derived from the epidermal mucus of Gram-positive and Gram-negative bacteria, multidrug resistance bacteria hagfish, with lysine, arginine, and tryptophan. Moreover, these peptides strains and did not cause significant lysis of human red blood cells and showed high binding affinity for both lipopolysaccharides and lipoteichoic were not cytotoxic to normal keratinocytes. The CD spectra of MAC acids and inhibited biofilm formation by most bacteria, but did not cause measured in the presence of trifluoroethanol (TFE) and sodium dodecyl significant lysis of human red blood cells and were not cytotoxic to normal sulfate (SDS) showed a high content α-helices. Furthermore, results of human keratinocytes. In addition, bacterial killing and membrane permeation NPN uptake assay and scanning electron microscopy (SEM) indicated that experiments demonstrated that the myxinidin analogs permeated through peptide killed microbial cells by increasing membrane permeability and bacterial membranes, leading to cytoplasmic disruption and cell death. Taken damaging membrane. Collectively, the results suggest that peptide have together, these findings suggest myxinidin analogs may be promising candidate potential for use as novel antimicrobial agents. antibiotic agents for therapeutic application against antibiotic-resistant bacteria.

38 | www.msk.or.kr Poster

D025 D027 Efficacy of Antimicrobial Peptide on Antibacterial, Antibacterial and Anti-inflammatory Effects of Novel P-AMP Anti-biofilm Activity against Human Pathogens

Jong Gwan Park1,2 and Yoon Kyung Park2* Na hee Kang1,2 and Yoonkyung Park1,2* 1Department of Convergence Science, Kongju National University 1Research Center for Proteineous Materials (RCPM), 2Department of Biotechnology 2Research Center for Proteineous Materials (RCPM), Chosun University and BK21-Plus Research Team for Bioactive Control Technology, Chosun University Dental caries and periodontitis are common bacterial mouth infections. Oral biofilms are multispecies microbial communities that exhibit high Numerous antimicrobial peptides (AMPs) from natural substance have resistance to antibiotics. Antimicrobial peptides (AMP) are emerging as been identified, isolated and characterized. We evaluated antimicrobial promising antimicrobial agents due to their rapid bactericidal activites. activities of P-AMP, a novel antimicrobial peptide, in vitro. The novel This study describes AMPs exhibits a broad spectrum of bactericidal peptide shows strong antimicrobial activity against Gram-negative and activity with eradicating capability on oral pathogens and respective biofilm. It Gram-positive bacteria but also exhibits no cytotoxicity toward mammalian is also stable in saline solution, saliva, brain-heart infusion medium. We cells. P-AMP appeared to act in the bacterial membrane. In cytotoxicity further compared the antimicrobial activity kinetics of AMP with chlorhexidine. assays, P-AMP showed no cytotoxicity toward RAW 264.7. Additionally, Flow cytometry was used to test if antimicrobial peptide can permeabilize binding of lipopolysaccharide to macrophages results in pro-inflammatory the membranes of oral pathogens. a significant proportion of oral pathogens cytokine secretion. Then, P-AMP has strong lipopolysaccharide neutralizing treated with antimicrobial peptide displayed propidium iodide fluorescent activity in RAW 264.7 cells exposed to LPS of P. aeruginosa. The peptides signal and the number of the bacterial cells with fluorescent signal increased were investigated for their ability to inhibit LPS-mediated cytokine release with the dose dependent manner. Additionally, these did not cause lysis of from RAW264.7 to bind LPS in solution, and when LPS is already bound to human red blood cell and were non cytotoxic to KB, pulp cell called macrophages. Finally, we conclude that P-AMP is an antimicrobial and epithelial cell and ondontoblast. This study provides a proof of concept in anti-endotoxin peptide that could serve as the basis for the development applying antimicrobial peptides in the clinical perspective. 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.

D026 D028 Antibacterial and Anti-inflammatory Activities of CMA3 Immunization of Mice with Irradiated Whole Cell Vaccine Peptide with Low Cytotoxicity in Escherichia coli Confers a Significant Degree of Protection against a Lethal Infection of Streptococcus agalactiae Jong-Kook Lee1 and Yoonkyung Park1,2* 1Research Center for Proteinaceous Materials (RCPM), 2Department of Biotechnology A-Yeung Jang, Yong Zhi, Bum Joo Kim, Zhao Lei, Zhang Jing, and BK21 Research Team for Protein Activity Control, Chosun University Shunmei Lin, and Ho Seong Seo* Radiation Biotechnology Division, Korea Atomic Energy Research Institute CA-MA is a hybrid antimicrobial peptide (AMP) derived from two naturally occurring AMPs, cecropin A and magainin 2. CA-MA shows strong antimicrobial The Gram-positive bacterium group B Streptococcus (GBS), known as activity against Gram-negative and Gram-positive bacteria but also exhibits Streptococcus agalactiae, is the most common cause of meningitis in the cytotoxicity toward mammalian cells. Our objective was to identify CA-MA newborns. It is also increasingly associated with invasive disease in non- analogues with reduced cytotoxicity by systematic replacement of amino pregnant adults, especially among the elderly and individuals with underlying acids with positively charged R groups, aliphatic R groups, or polar R chronic illnesses. Since GBS whole cell vaccine (WCV) is expected to confer groups. CMA3 appeared to act by inducing pore formation (toroidal protection against GBS infection, we investigated the efficacy of irradiated model) in the bacterial membrane. In cytotoxicity assays, CMA3 showed little killed WCV as compared to chemically killed WCV. GBS WCV was prepared cytotoxicity toward human red blood cells or HaCaT cells. Additionally, no by treating gamma-irradiation at 9kGy/hr or formalin for 2hr. ICR mice fluorescence was released from small or giant unilamellar vesicles exposed were immunized through intraperitoneal injection. Although irradiated μ to 60 M CMA3 for 80 s, whereas fluorescence was released within 35 s WCV increased anti-GBS Ab titer in serum higher than level to chemical upon exposure to CA-MA. CMA3 also exerted strong lipopolysaccharide- WCV, it was elicited significantly higher rate of survival in GBS infection neutralizing activity in RAW 264.7 cells, and BALB/c mice exposed to LPS model. Also, irradiated WCV-vaccinated mice were increased Ab titer to after infection by Escherichia coli showed improved survival after administration other serotype GBS strains compare with chemical WCV. Moreover, splenocytes of one 0.5-mg/kg of body weight or 1-mg/kg dose of CMA3. Finally, in a isolated from irradiated WCV-vaccinated mice were increased population mouse model of septic shock, CMA3 reduced the levels of proinflammatory of CD3+ CD4+ T cells than chemical WCV and it was increased level of factors, including both nitric oxide and white blood cells, and correspondingly CD4+ T cell-associated cytokine (TNF-α, IL-6 and IL-10) with chemical reduced lung tissue damage. This study suggests that CMA3 is an antimicrobial/ WCV. These findings suggest that irradiated WCV elicit effective response antiendotoxin peptide that could serve as the basis for the development of CD3+ CD4+ T cells that protects GBS infection. of anti-inflammatory and/or antimicrobial agents with low cytotoxicity.

www.msk.or.kr | 39 2016 International Meeting of the Microbiological Society of Korea

D029 D032 Pathogenicity of Newly Isolated Human Ileal Streptococci Chitosan Nanoparticle Supplemented Diet Alters the Zebrafish Gut Microbiota 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* Chathurica Udayangani, Sajith Dananjaya, Department of Life and Nanopharmaceutical Sciences and Department of Seung Beom Seo, and Mahanama De Zoysa* Biology, Kyung Hee University College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungnam National University 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 This study was conducted to investigate the effects of dietary CNP on the cancer. The isolates were assigned to the members in mitis group streptococci gut microbial communities of zebrafish. Fish diet with CNPs was prepared and identified as novel species closely related with S. parasanguinis and S. by adding 2% of CNP to the commercial feed on a weight basis. Zebrafish australis based on polyphasic analyses. Thus, we designate the two isolates (Danio rerio) were maintained (12 fish/tank) for 4 weeks and fecal matter as Streptococcus ilei. To assess abundance and incidence of S. ilei in was collected. 16S rRNA gene sequence based metagenomics sequencing human body sites, we estimated S. ilei frequencies in various human body was conducted to understand the change of microbiome profiles. Total of sites using metagenomic data (506 experiments, 6,228 samples and 56 and 53 operational taxonomic units (OTUs) were resulted for control 22,875,147 reads) from Human Microbiome Project (HMP) database with and CNP treated groups, respectively. Upon CNP treatment considerable matching S. ilei specific sequence. S. ilei occurs with 0.18 to 2.09% relative changes in the natural gut bacterial composition was observed. In both abundance and 19 to 86.98% incidence depending on each body site. To control and CNP treatments, phylum Proteobacteria was found to be the identify pathogenic potential of S. ilei, cytotoxicity of S. ilei was ascertained most abundant in zebrafish gut as reported in previous studies. However, by MTT assay on A549 and HT-29 cell lines. S. ilei and its supernatant show 19% reduction in phylum Proteobacteria and 82.02% increase in phylum cytotoxic response to A549 and HT-29 cell lines as similar as pathogenic S. Fusobacteria were observed in the fish fed with CNP supplemented pneumoniae. Furthermore, exposures of S. ilei via intraperitoneal routes diet. Even though the exact function of certain microbial species are yet to causes mortality in immunocompromised mice and healthy mice models. be discovered, the results suggest that CNP could cause beneficial modifications Thus, these results disclose presence of S. ilei in human microbiota and of gut microbiome to enhance the digestibility and immunity of fish. demonstrate its pathogenic potential. [Granted by Mid-career Researcher Program (2012-0008806) through the Keywords: Chitosan nano-particles (CNP); gut; Metagenomics; Proteobacteria; National Research Foundation of Korea.] Fusobacteria; zebrafish [This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2014R1A2A1A- 11054585).]

D030 D033 Prevalence of Respiratory Viruses in Patients with Acute Fusarium oxysporum Infestation of Zebrafish in Laboratory Respiratory Infections in Jeonbuk, 2015 System

1 1 1 1 Chan mun Jin , Yeun Jeong Kim , Keung Eu No , Seouk Hyeon Lim , Sang Yeop Shin, Chanuka Kulatunga, Dong Joon Kim, 1 2 2 Cheon Hyeon Kim , Hee Dong Jung , Hyang Min Cheong , Bae Keun Park, and Mahanama De Zoysa* 2 2 Sung Soon Kim , and Ki Soon Kim * College of Veterinary Medicine and Research Institute of Veterinary Medicine, 1 2 Jeollabukdo Institute of Health and Environment Research, Center for Chungnam National University Infectious diseases, KNIH, KCDC Fusarium oxysporum is a highly diverse fungi inhabited in every ecosystem In this study, to investigate the viral pathogen causing Acute Respiratory as a saprophyte or a pathogenic fungi. Recently, unusual F. oxysporum Infections (ARI), multiplex PCR/RT-PCR was performed on respiratory specimens infestation was identified from zebrafish (Danio rerio) culture system. (throat or nasal swab) collected from the ARI patients. And the statistical Initially, rapid whitish smudge was appeared in the water with the severity, analysis was performed to investigate the characteristics of age distribution, fungal blooming on fish tank walls, clogging of water circulatory tubes was seasonality, and clinical features of ARI patients with respiratory virus observed. Reduction of fish vigor and fungal gill infection of zebrafish was infection in Jeonbuk, 2015. noticed in the affected water systems. Fungal isolation was done by inoculating We performed multiplex PCR/RT-PCR on respiratory specimens to determine the specimens on potato dextrose agar (PDA) at room temperature. Microscopic the prevalence of 16 viruses including adenovirus(HAdV), parainfluenza studies on fungal hyphae in the water system and the PDA, colony pigmentation, virus(HPIV) 1, 2, 3, respiratory syncytial virus(HRSV) A and B, influenza clamidospore formation and the presence of macro and microspores were virus (IFV) H1N1, H3N2, B, human coronavirus(HCoV) 229E, NL63 and OC43, morphologically confirmed the fungi as the F. oxysporum. Furthermore, human rhinovirus(HRV), human bocavirus(HBoV), human metapneumovirus the internal transcribed spacer regions of nuclear rDNA repeat units (ITS) (HMPV) and Enterovirus(HEV). Respiratory viruses were detected by sequences of isolated fungi were identical to nine F. oxysporum sequences 51.3% of enrolled patients (n=779) in 2015. The most common respiratory in Genbank. Experimental hypodermis injection of zebrafish with F. oxysporum virus isolated was influenzavirus 82 cases (23.4%) followed by rhinovirus was developed the infection by revealing the virulence of these isolates. 145 cases (41.3%), enterovirus 49 cases (6.3%), adenovirus 44 cases (12.5%), Histopathological studies were carried out to examine the pathogen parainfluenzavirus 33 cases (9.4%), coronavirus 18 cases (5.1%), metapneumovirus invasion in zebrafish tissues. 12 cases (3.4%), respiratory syncytial virus 10 cases (2.8%) and bocavirus 7 cases (2.0%). Keywords: Danio rerio; Fusarium oxysporum; infestation; fungal hyphae A large proportion of respiratory infections in the community in Jeonbuk [This work was supported by a National Research Foundation of Korea was associated to viruses. HRSV, IFV, and HCoV infections peaked on (NRF) grant funded by the Korea government (MSIP) (2014R1A2A1A- winter season, in summer, HEV were epidemic. 11054585).] [Supported by grants from KCDC]

40 | www.msk.or.kr Poster

D034 D036 Antibiotic Resistance is Induced by a Bacterial Starvation Identification of Essential Genes of Pseudomonas Signal in Vibrio cholerae aeruginosa for It Is Growth in Airway Mucus

Hwa Young Kim, Young Taek Oh, and Sang Sun Yoon* Mohammed Mohammed and Sang Sun Yoon* Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, Institute for Immunology and Immunological Diseases, Yonsei Medical Science, Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine University College of Medicine

When bacteria encounters variety of growth-inhibitory stresses, the stringent Pseudomonas aeruginosa has been identified as an important causative response (SR) is activated by accumulation of (p)ppGpp, a small nucleotide agent for airway infection. Mucin is a major component of airway mucus regulator known as a stress alarmone. Accumulation of (p)ppGpp results and is heavily O-glycosylated with a protein backbone. Airway infection is in a profound reprogramming of global gene expression. Here, we show known to be established with bacterial adhesion to mucin. However, genes that the bacterial capability to produce (p)ppGpp is involved in regulating involved in mucin degradation or utilization still remains elusive. In this antibiotic resistance in V. cholerae. N16961, a 7th pandemic V. cholerae study, we thought to provide a genetic basis of P. aeruginosa airway growth strain, became resistant to tetracycline, when (p)ppGpp accumulation by identifying those genes. First, we compared genome-wide expression was induced. N16961 and ΔrelAΔspoT, a (p)ppGpp-overproducing mutant profiles of PAO1, a prototype strain, grown in M9-mucin (M9M) and strain, were more resistant to the treatment of lethal concentration of M9-glucose (M9G) media by RNASeq analyses. PA2939 and PA0122 genes tetracycline, when compared with (p)ppGpp0 ((p)ppGpp-null mutant). Similar encoding putative aminopeptidase and a hemolysin homologue were effects were also induced, in response to the treatment with other among top 5 genes, whose expression were increased >100-fold in M9M-grown antibiotics, such as erythromycin and chloramphenicol. Furthermore, the PAO1. Second, a PAO1 transposon (Tn) insertion mutant library was ΔrelAΔspoT mutant, which mounted resistance, lost typical curved-rod screened for mutants defective in growth in M9M media. Six mutants with shape morphology and looked smaller in size based on scanning electron Tn insertion were determined to exhibit faulty growth in M9M. These genes microscope analysis. N16961, when harvested at stationary phase, also are involved in the synthesis of leucine and tryptophan. Importantly, all of exhibited similar cell shape change, while (p)ppGpp0 mutant maintained these mutants were incapable of establishing mouse airway infection, their normal cell shape. We also found that N16961 gained smaller cell suggesting that these gene functions are required for P. aeruginosa in vivo shape phenotype, when (p)ppGpp accumulation was induced by serine infectivity. Further mechanistic dissection of this particular process will hydroxamate at exponential phase. Together, our results demonstrate reveal new drug targets, inhibition of which could control recalcitrant P. that the alteration of bacterial cell phenotype by accumulation of intracellular aeruginosa infections. (p)ppGpp is associated with antibiotic resistance in V. cholerae.

D035 D037 Structural Insight of Substrate Promiscuity for Anti-tumor Effect of Quercetin in EBV-associated Human 2-deoxyribose-5-phosphate Aldolase (DERA) from Gastric Carcinoma Streptococcus suis Hwan Hee Lee, Seulki Lee, Hyojeong Kang, and Hyosun Cho* Thinh-Phat Cao1, Joong-Su Kim2, and Sung Haeng Lee1* Department of Pharmacy, Duksung Women’s University 1Department of Cellular and Molecular Medicine, Chosun University School of Medicine, 2Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Licorice extracts have been widely used in herbal and folk medications. Biotechnology Glycyrrhiza yields diverse range of biological compounds including triterpene (glycyrrhizin, glycyrrhizic acid) and flavonoids (quercetin, liquiritin, liquiritigenin, 2-deoxyribose-5-phosphate aldolase (DERA) belongs to a class I aldolase glabridin, licoricidin, isoliquiritigenin). The flavonoids of licorice are known catalyzing aldol condensation of two aldehydes in the active site, and is to have strong activities against cancer. Quercetin, the most abundant particularly intersting target to drug manufacture. Structural and biochemical compound in flavonoids, has been shown to perform anti-ulcer, anti-cancer, studies have shown that the active site of DERA is typically flexible and antioxidant and anti-inflammatory properties. displays broader substrate specificity. The most distinctive structural feature Latent infection of EBV leads to serious malignancies such as Burkitt’s of DERA is short and flexible C-terminal region, which is also responsible lymphoma, Hodgkin’s disease and gastric carcinoma (GC). In fact, EBVaGC for substrate recognition. Therefore, substrate tolerance may be related is known to be one of the most common cancers among EBV associated to the C-terminal structural features of DERA. Here, we determined the tumors. crystal structures of full length and C-terminal truncated DERA from In this study, we first examined anti-cancer effect of quercetin and Streptococcus suis (SsDERA). In common, both contained the typical (α/ isoliquiritigenin in vivo xenograft mouse models implanted with EBV negative β)8 TIM-barrel fold of class I aldolases. Interestingly, C-terminal truncation carcinoma (MKN74 cells) as well as EBV positive carcinoma (SNU719 cells). resulting in missing the last α9 and β8 secondary elements, allowed DERA Secondary, we explored the molecular mechanisms responsible for the to maintain activity comparable to the full-length enzyme. Typically, Arg186 anti-viral and anti-cancer activities. and Ser205 residues at the C-terminus appeared mutually supplemental The results showed that quercetin and isoliquiritigenin have the effect of or less indispensible for substrate phosphate moiety recognition. Our results anti-tumor in mice injected with or without EBV. Also, our results indicated suggest that DERA might adopt a shorter C-terminal region than conventional that quercetin affects the expression of p53-associated proteins including aldolases during evolution pathway, resulting in a broader range of substrate p21, caspases and Bcl-2 families as well as the expression of EBNA-1, tolerance through active site flexibility. BZLF-1, and LMP-1 proteins in tumor tissue specimen of mice injected gastric carcinoma of presence or absence with EBV.

www.msk.or.kr | 41 2016 International Meeting of the Microbiological Society of Korea

D038 D040 The Anti-HSV Effect of Quercetin in Raw 264.7 Cells via Significant Differences between Tick Bite Sites and Mite Bite Downregulation of Inflammatory Responses Sites on Humans

Seulki Lee, Hwan Hee Lee, and Hyosun Cho* Choon-Mee Kim1, Na-Ra Yoon2, and Dong-Min Kim2* Department of Pharmacy, Duksung Women’s University 1Premedical Science, College of Medicine, Chosun University, 2Department of Internal Medicine, College of Medicine, Chosun University Quercetin is one of major ingredients derived from Glycyrrhiza uralensis, which is largely used as traditional medicine in Asian countries. Quercetin Identification of mite and tick bite sites provides an important clinical clue was reported to have several biological activities including anti-viral effect for diagnosis. To date, there has been no comparative study on preferred as well as anti-inflammatory effect. We explored the molecular mechanism bite sites between mites and ticks in patients. This study was conducted by that links anti-viral and anti-inflammatory activities using HSV infection reviewing medical records and through a field study. For mite bite sites, system. eschars were checked on 506 patients with scrub typhus, as confirmed To identify the downregulation of inflammatory response, raw 264.7 cells using immunofluorescence assay or nested polymerase chain reaction were treated with various concentrations of quercetin with or without targeting the 56-kDa gene. Tick bite sites were identified and marked on a HSV infection for 24 h and cell pellet (or supernatant) was collected. Then, diagram for 91 patients who experienced tick bites within the previous we performed HSV plaque assay and ELISA assay. Next, we measured the year through a field epidemiological investigation. The mite bite and tick expressions of HSV proteins (ICP0 and gD), NFkB and TLRs associated with bite sites were compared. The most frequently observed mite bite sites anti-inflammatory genes using qPCR and Western blot analysis. were the anterior chest, including the axillary region (29.1%), and abdominal Quercetin significantly decreased HSV infectivity in raw 264.7 cells through region, including the inguinal area (26.1%). Tick bite sites were most frequently HSV plaque assay, and also suppressed the production of inflammatory found on the lower extremities (33.0%), followed by the abdominal region, cytokines (IL-1β and TNF-α). In addition, quercetin decreased the expression including the inguinal area (26.4%), and upper extremities (26.4%). The of HSV proteins (ICP0 and gD) and inflammatory genes including NFkB and distribution was significantly different between mite bite and tick bite TLR3 in raw264.7 cells. sites (p<0.001). There was a statistically significant difference in the mite Therefore, we suggest that the anti-viral effect of quercetin was directly bite sites (p=0.001) but not the tick bite sites (p=0.985) between men and correlated with anti- inflammatory responses in HSV-infected raw 264.7 women. cells. 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.

D039 D041 KSHV Infection of Primary Human Endothelial Cells Induces Utility of Tick-bite Site Samples for the Diagnosis of Human Complement Activation by Exosome-mediated Binding of Granulocytic Anaplasmosis Properdin Choon-Mee Kim1 and Dong-Min Kim2* 1 1 1 Hyungtaek Jeon , Jisu Lee , Seung-min Yoo , 1Premedical Science, College of Medicine, Chosun University, 2Department of 2 1 Shou-Jiang Gao , and Myung-Shin Lee * Internal Medicine, College of Medicine, Chosun University 1Department of Microbiology and Immunology, Eulji University School of Medicine, 2Department of Molecular Microbiology and Immunology, Keck School of Medicine, Human granulocytic anaplasmosis (HGA) is a tick-borne infectious disease University of Southern California, Los Angeles, California, USA caused by Anaplasma phagocytophilum, an obligate intracellular bacterium. Until now, the utility of tick-bite site samples for HGA diagnosis has not The complement system is an important component of immune defense been reported. Using a patient’s buffy coat and tick-bite site crust, we against pathogens. Physiologically, complement system can induce inflammation, performed polymerase chain reaction (PCR) testing using Ehrlichia or opsonization, and MAC-mediated cell lysis. In addition to the roles in a first Anaplasma-specific primers. PCR with Anaplasma-specific primers and defense system, complement participates in diverse immunological processes buffy coat and crust specimens obtained before doxycycline administration including control of adaptive immunity, removal of apoptotic cells, angio- was positive, whereas PCR with buffy coat specimens obtained six days genesis, etc. Inflammation is one of pathologic features of Kaposi's sarcoma but after doxycycline administration was negative. However, PCR with tick-bite little has been known for the activation of complement system in KSHV. We site crust specimens obtained six days after doxycycline administration have reported complement activation on latent KSHV-infected cells, providing remained positive. Therefore, like buffy coat PCR, PCR with a tick-bite site survival signals for the KSHV-infected cells. Here, we aimed to investigate crust appeared useful in the early diagnosis of HGA. This is the first study whether complement system is activated during acute KSHV-infection in to suggest that tick-bite site samples are useful for HGA diagnosis in patients HUVECs. Despite several complement regulatory proteins were highly who have already been treated with antibiotics such as doxycycline. 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.]

42 | www.msk.or.kr Poster

D042 D044 Clinical Usefulness of Conventional PCR Targeting the 16S Conditionally Pathogenic Gut Microbes Promote Larval Ribosomal RNA for the Diagnosis of Scrub Typhus Growth by Increasing Redox-Dependent Fat Storage in High Sugar Diet-Fed Drosophila melanogaster Choon-Mee Kim1 and Dong-Min Kim2* 1Premedical Science, College of Medicine, Chosun University, 2Department of Tae Woong Whon, Na-Ri Shin, Mi-Ja Jung, Dong-Wook Hyun, Hyun Sik Kim, Internal Medicine, College of Medicine, Chosun University Pil Soo Kim, and Jin-Woo Bae* Department of Life and Nanopharmaceutical Sciences and Department of Biology, Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi. Kyung Hee University Delays in diagnosis and antibiotic administration can cause fatal complications. Therefore, a rapid and early diagnosis is critical. Changes in the composition of the gut microbiota contribute to the We retrospectively evaluated the accuracy of conventional PCR targeting development of obesity and subsequent complications associated with the 16S rRNA gene (16S C-PCR) using the Ot-16sRF1/Ot-16sRR1 primers metabolic syndrome. However, the role of increased numbers of certain for the diagnosis of scrub typhus. We examined blood specimens from bacterial species during the progress of obesity remains unclear. Here, we 115 adult patients confirmed to have scrub typhus, 52 patients confirmed show that chronic feeding of a high sugar diet (HSD) to Drosophila melanogaster to have characterized illness and 35 febrile patients who had neither scrub resulted in a predominance of resident uracil-secreting bacteria in the gut. typhus nor any characterized illness. To evaluate the clinical usefulness of Axenic insects mono-associated with uracil-secreting bacteria or supplemented this new method, we compared its diagnostic effectiveness with that of with uracil under HSD conditions promoted larval development. Redox the following methods: C-PCR targeting the 47-kDa, 56-kDa, and groEL signaling induced by bacterial uracil promoted larval growth by regulating genes; nested PCR targeting the 47-kDa and 56-kDa genes; and real-time sugar and lipid metabolism via activation of p38a mitogen-activated protein PCR targeting the 47-kDa gene. kinase. The present study identified a new redox-dependent mechanism The C-PCR with the Ot-16sRF1/Ot-16sRR1 primers detected O. tsutsugamushi by which uracil-secreting bacteria (previously regarded as opportunistic infection with a sensitivity of 87% and a specificity of 100%. Other C-PCR pathobionts) protect the host from metabolic perturbation. Thus, changes amplified the 47-kDa, 56-kDa, and groEL genes with lower sensitivities of in the gut microbiota play an important role in alleviating deleterious 3%, 8%, and 66%, respectively. Nested PCR amplification of the 47-kDa diet-derived effects such as hyperglycemia. and 56-kDa genes showed sensitivities of 81% and 80%, respectively, [Supported by a grant from the Mid-career Researcher Program (2011- whereas real-time PCR of the 47-kDa gene exhibited a sensitivity of 76%. 0028854 to J.-W.B.) through the National Research Foundation of Korea The 16S C-PCR using the Ot-16sRF1/Ot-16sRR1 primers is simple, clinically (NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP), and useful. from the Basic Science Research Program (2015019390 to T.W.W.) through the NRF funded by the Ministry of Education.]

D043 D045 Host Transcriptional Profiles of Acinetobacter Phylogenetic Analysis of Leptospira spp. in Wild Rodent at baumannii-Infected Human Lung Cancer Cell Gwangju Metropolitan Area, Republic of Korea 2014~2015

Sang-Yeop Lee1, Edmond Changkyun Park1,2, Sung Ho Yun1, Jung Wook Park1, Sun Hee Kim1, Duck Woong Park1, Hye Jung Park1, So Chi Won Choi1, Hayoung Lee1, Gun-Hwa Kim1,3, and Seung Il Kim1* Hyang Jeong1, Mi Hee Seo1, Yong Seok Lee1, Jae Keun Chung1, Hyun Jae 2 2 3 1Division of Bioconvergence Analysis, Korea Basic Science Institute, 2Department Song , and Jung Yoon Lee , and Dong Min Kim * of Bio-Analytical Science, University of Science and Technology, 3Department of 1Health and Environment Institute of Gwangju, 2Gwangju Health University Functional Genomics, University of Science and Technology 3Chosun University, Medical College

Multidrug resistant Acinetobacter baumannii is a predominant causative Leptospirosis is an important zoonotic disease worldwide, acquired through organism of pneumonia by hospital outbreaks. However, details of infection direct contact with animal reservoirs and an environment contaminated mechanism of A. baumannii were not elucidated well. To investigate host by their urine. To obtain a better understanding of prevalence in wild rodents response by A. baumannii infection, A549 lung cancer cells were treated at Gwangju metropolitan area, we conduct a survey from September, with whole bacterial cells, membrane fraction, or outer membrane vesicles 2014 to December, 2015. of A. baumannii and transcriptomic signature of the A549 cells were Wild rodents were monthly captured by sherman live trap. Kidney specimens analyzed. As a result, whole cell-infected A549 cells were differentially of 238 wild rodents from 2 area were examined by PCR based 3 primer expressed 2,324 genes compared to untreated control A549 cells. Most of (flaB, secY, 16srRNA). Leptospiral DNA was amplified in 21.8% (52/238) those genes were related to biological process of cell cycle, apoptosis and including 5.9% (14/238) in flaB primer, 8.8% (21/238) in secY primer and cell death. Among them, Jun, GADD45A and IL8 were highly expressed in 7.1% (17/238) in 16S rRNA. Of them, 17 sequences (32.7%; 17/52) were A. baumannii-infected A549 cells. Jun and GADD45A were commonly involved analysed. They had a homology with Leptospira spp. of GenBank. in mapk signaling pathway. In A. baumannii membrane factions-treated Our results provides a baseline data for Leptospirosis in Gwangju metropolitan A549 cells, 1,039 genes were differentially expressed comparing to untreated area. To predict reemerging of this disease in human, needs continuous control A549 cells. Those genes were mainly participated in the process of wildlife monitoring cell cycle arrest and inflammation response. In A549 cells treated with A. [Suppoted by Healh & Environment Research institute of Gwangju city.] 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.

www.msk.or.kr | 43 2016 International Meeting of the Microbiological Society of Korea

D046 D048 Aggregatibacter actinomycetemcomitans Lipopolysaccharide- Acute Gastroenteritis Surveillance from Diarrheal Patients mediated Induction of Chemokines MCP-1, MIP-1α, and in Gwangju, Korea, During 2015 IP-10 Occurs via Distinct Intracellular Signaling Pathways in Seon Kyeong Kim, Hye-young Kee, Tae Sun Kim, Eun-hye Jo, Ji Hyun Shin, Murine Macrophages Dong Ryong Ha, Eun-Sun Kim, and Kye Won Seo* Health & Environment Institute of Gwangju Ok-Jin Park, Min-Kyung Cho, Cheol-Heui Yun, and Seung Hyun Han* Department of Oral Microbiology and Immunology, School of Dentistry, Seoul Acute gastroenteritis (AGE) has been recognized as one of the most National University common diseases in humans and continues to be a major public health problem worldwide, primarily occurs in infants and young children in both Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium and developed developing countries. A total of 1,525 samples of rectal frequently isolated from lesions of patients with localized aggressive swabs and stool specimens of diarrheal patients were collected from 9 periodontitis. Lipopolysaccharide (LPS), a major cell wall component of pediatric hospitals and internal medicine during 2015. For detection of 10 Gram-negative bacteria, stimulates innate immune cells via Toll-like receptor species bacterial pathogens including salmonella and 5 enteric virus including 4 (TLR4) to initiate inflammatory responses. In this study, we purified LPS norovirus, We analyzed by the isolation culture, biochemical identification from A. actinomycetemcomitans (AaLPS) and investigated its ability to and enzyme immunoassay, reverse transcription-PCR, DNA sequencing. induce the expression of chemokines. AaLPS induced the expression of Enteric viruses and bacteria were detected in 447 (29.3%) and 393 (25.8%) chemokines, MCP-1, MIP-1α, and IP-10, leading to the infiltration of peripheral specimens, respectively. Among the enteric viruses detected, norovirus blood mononuclear cells in a transwell system. Although TLR4 was essential 305 (20.0%) and rotavirus 88 (5.8%) were the predominant causative for the induction of all these chemokines by AaLPS, MCP-1 and MIP-1α agents, followed by astrovirus 28 (1.8%), adenovirus 19 (1.3%) and sapovirus 7 expressions were MyD88-dependent, but IP-10 expression was MyD88-independent. (0.5%). Among the pathogenic bacteria, Salmonella accounting for 115 Furthermore, the activation of ERK and JNK were necessary for the expression (7.5%) was the most frequent pathogen, followed by Staphylococcus of MCP-1 and MIP-1α, whereas p38 MAP kinase and JNK activations were aureus accounting for 108 (7.1%), pathogenic E. coli accounting for 88 required for IP-10 expression. In addition, IFN-β/STAT1 signaling was (5.7%). The epidemic peaks of the enteric viruses were October to January exclusively involved in IP-10 expression but not in MCP-1 or MIP-1α for norovirus, January to April for rotavirus. The seasonal activity of expression. AaLPS also activated NF-κB, AP-1, NF-IL6, and ISRE, all of rotavirus was shifted from winter to late spring. Sequencing of the which are involved in chemokine gene expression. These results suggest norovirus strains revealed that GII.4/2012 (80/305, 26.2%) sydney variant that AaLPS induces the expression of chemokines MCP-1, MIP-1α, and was the most common genotype and followed by GII.3 (35/305, 11.5%) IP-10 through TLR4. Further, the induction of MCP-1 and MIP-1α requires and GII.17 Kawasaki (28/305, 9.2%) strains. MyD88, ERK, and JNK, whereas the induction of IP-10 requires JNK, p38 Ongoing surveillance will help further elucidate trends, identify gaps, and MAP kinase, and IFN-β/STAT1. assess the effects of future interventions on reducing epidemic gastroenteritis. D047 D049 Acquisition of Chemoresistance and Other ɩNKT Cell Sensitization during Neonatal Respiratory Syncytial Malignancy-related Features of Colorectal Cancer Cells are Virus Infection Induces Severe Pulmonary Pathology in Incremented by Ribotoxic Mycotoxin and Antibiotics Re-infected Adult Mice

1 2 3 Chang-Kyu Oh , Dongwook Kim , Seung-Joon Lee , Seung Young Lee1, Youran Noh1, Semi Rho1, Jung Hyun Goo1, 3 3 Seong-Hwan Park , and Yuseok Moon * Min Jung Kim1, Chang-Yuil Kang2, Man Ki Song1, and Jae-Ouk Kim1* 1 Department of Biomedical Sciences, Pusan National University School of Medicine 1Laboratory Science, International Vaccine Institute 2 National Institute of Animal Science, RDA 2College of Pharmacy, Seoul National University 3Department of Biomedical Sciences, Pusan National University Respiratory syncytial virus (RSV) is a major viral pathogen that causes Colorectal cancer (CRC) as an environmental disease is largely influenced severe lower respiratory tract infections in human infants worldwide. Infants by accumulated epithelial stress from diverse environmental causes. We with severe RSV bronchiolitis tend to experience more wheezing and are exposed to ribosome-related insults including ribosome-inactivating asthma in later childhood. Because invariant natural killer T (ιNKT) cells stress (RIS) from mycotoxins and antibiotics, but their physiological impacts are associated with the pathology of asthma, we investigated whether on the chemotherapy of CRC are not yet understood. Here, we revealed neonatal ιNKT cells are involved in aggravation of pulmonary diseases following the effects of RIS on chemosensitivity and other malignancy-related properties RSV infection. Intranasal exposure of the ιNKT cell ligand, α-galactosylceramide of CRC cells. First, RIS led to bidirectional inhibition of p53-macrophage (α-GC), with RSV primary infection in neonatal mice elicited neither cytokine inhibitory cytokine 1 (MIC-1)-mediated death responses in response to production nor pulmonary eosinophilia despite of the presence of both anticancer drugs by either enhancing ATF3-linked antiapoptotic signaling CD1d+ cells and ιNKT cells. Interestingly, in adult mice re-infected with or intrinsically inhibiting MIC-1 and p53 expressions, regardless of ATF3. RSV, neonatal ιNKT cell sensitization by α-GC during RSV primary infection Second, RIS enhanced epithelial mesenchymal transition (EMT) and biogenesis resulted in much higher levels of pulmonary Th2 cytokines and elevated of cancer stem-like cells (CSC) in an ATF3-dependent manner. These findings eosinophilia with severe airway hyperresponsiveness. In contrast, α-GC indicated that gastrointestinal exposure to RIS interferes with the efficacy priming of adults during RSV re-infection did not induce more severe of chemotherapeutics, mechanistically implicating that ATF3-linked malignancy airway symptoms than RSV re-infection in the absence of α-GC. α-GC and chemoresistance can be novel therapeutic targets for treatment of co-administration during RSV primary infection facilitates RSV clearance environmentally aggravated cancers. regardless of age, but the viral clearance following re-infection was not ι [This work was carried out with the support of the Cooperative Research NKT cell-dependent. This study suggests that neonatal ιNKT cell sensitization Program for Agriculture, Science & Technology Development (Project No. during RSV primary infection is strongly associated with exacerbation of PJ01093206)" Rural Development Administration, Republic of Korea).] pulmonary diseases following RSV re-infections in adulthood. [Supported by KNRF (NRF-2014R1A1A3049897)]

44 | www.msk.or.kr Poster

D050 D052 B Cell Infection by Kaposi’s Sarcoma-associated Herpesvirus Human Fucosyltransferase 2 Expression in Mouse

Jinjong Myoung Jinjong Myoung Korea Zoonosis Research Institute, Chonbuk National University Korea Zoonosis Research Institute, Chonbuk National University

Kaposi’s sarcoma-associated herpesvirus is known to infect B cells and Human norovirus (hNoV) accounts for over 90% of virus-mediated acute endothelial cells. Among them, B cells seem to serve as the main virus gastroenteritis, thus posing a serious threat to public health. It was first reservoir and migration vector in Kaposi’s sarcoma. KSHV infection in B identified in 1968, however, due to the lack of susceptible cell lines and cells induces two very different B cell lymphomas: multicentric Castleman’s animal model, progress in its pathophysiology has been serious hindered. disease (MCD) and primary effusion lymphoma (PEL). However, interestingly, As human fucosyltransferase 2 (hFUT2) seems to be the disease determinant, peripheral blood B cells have been resistant to KSHV infection ex vivo, we have set out to develop hFUT2-transgenic mice as a small animal which deters detailed delineation of tumorigenesis mechanisms in B cells. model for hNoV. hFUT2-expressing transgenic mice were backcrossed to Here, we report that human lymphoid aggregate cultures (HLAC’s), established C57BL/6 and BALB/c background. When hFUT2-Tg with B6 background from tonsils, are susceptible to KSHV infection. Infected B cells display were inoculated with hNoV (1 × 106 genome copies/mouse), low levels of spontaneous lytic replication, thus more closely resembling the pathobiology viral replication was detected in various tissues, including liver, small of MCD. This in vitro model will undoubtedly provide a useful model for intestines, and spleen. Unfortunately, as backcrossing increases hFUT2-Tg studying KSHV biology in B cells. 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.

D051 D053 OX40 and 4-1BB Differentially Inhibit KSHV Replication in B KSHV Infection in Established B Cells Require Cell-associated Cells and Endothelial Cells Viruses

Jinjong Myoung Jinjong Myoung Korea Zoonosis Research Institute, Chonbuk National University Korea Zoonosis Research Institute, Chonbuk National University

OX40 belongs to the tumor necrosis factor superfamily, and is known to Kaposi’s sarcoma-associated herpesviral infection in primary B cells provides modulate T cell activation and memory T cell generation. Previously, Byun an exciting model for the investigation of KSHV biology in B cells. Ex vivo et al elegantly demonstrated genetic deficiency of OX40 leads to childhood infection of tonsillar B cells display a moderate level of survival advantage Kaposi’s sarcoma, underscoring the importance of OX40 co-stimulation in over uninfected B cells. However, infected tonsillar B cells are neither the generation of KSHV-specific T cell activation. Here, we report for the immortalized or transformed, which poses barriers to study biochemical first time that ligation of OX40L on endothelial cells with recombinant events in B cells upon KSHV infection. As such, many attempts had been OX40 effectively inhibits KSHV replication. Interestingly, previously unsuspected made to infect established lymphoblastoid cells by KSHV with no avail. Here, roles of 4-1BB in KSHV replication were highlighted in infected endothelial we report immortalized or transformed B cells are rendered susceptible to cells as well. On the contrary, in infected B cells, neutralization of 4-1BB- KSHV infection only when in direct contact with cell-associated viruses. 4-1BBL, but not that of OX40-OX40L, inhibits KSHV replication, indicating Upon infection, B cells are mostly latently infected, which may provide a differential role of OX40 in two different main targets of KSHV in vivo. model for investigating KSHV-mediated interventions of signal transduction pathways in B cells.

www.msk.or.kr | 45 2016 International Meeting of the Microbiological Society of Korea

D054 D056 Jak-STAT Pathway Enhances KSHV Replication in Endothelial Prooxidant Activity of Pyrogallol on Vibrio vulnificus Infection Cells Ju Young Lim and Young Ran Kim* Jinjong Myoung College of Pharmacy, Chonnam National University Korea Zoonosis Research Institute, Chonbuk National University Vibrio vulnificus, the causative agent of fatal septicemia and necrotic Since KSHV discovery in the early 1990’s, it has been known that IL-6 is the wound infection, is a good model organism for bacterial septicemia studies. tumor growth factor in KSHV-mediated malignancies. As IL-6 is the canonical We found that the polyphenol pyrogallol protected HeLa cells from repeats- activator of Jak-STAT3 pathway and STAT3 is an oncogene, it has been in-toxin A1 (RtxA1)-mediated morphological damage and V. vulnificus-induced assumed that IL-6 and Jak/STAT3 are involved in tumorigenesis, but not in cytotoxicity. Pyrogallol also inhibited the growth of V. vulnificus, and the viral replication. Here, we report that STAT3 is both necessary and required inhibitory effect was more significant in the log phase than in the stationary for efficient viral replication in endothelial cells. As in Kaposi’s sarcoma phase. To investigate the inhibitory mechanism, pyrogallol-induced toxicity was compared between a V. vulnificus catalase–peroxidase (katG−) and multicentric Castleman’s disease viral replication is required for tumor − growth, we propose that STAT3-mediated enhancement of KSHV replication mutant and the wild-type MO6-24/O strain. The katG mutant did not may be an important mechanisms for KSHV-mediated tumorigenesis. 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

D055 D057 Calcineurin Targets Involved in Stress Survival and Fungal Role of Host Cell Filamin A in Vibrio vulnificus RtxA1 Virulence Toxin-induced Cytoskeletal Rearrangement and Cytotoxicity

Hee-Soo Park1,2*, Joseph Heitman2, and Maria E. Cardenas2 Ju Young Lim and Young Ran Kim* 1School of Food Science and Biotechnology, Kyungpook National University, College of Pharmacy, Chonnam National University 2Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA Cytoskeletal rearrangement and acute cytotoxicity are observed in Vibrio vulnificus RtxA1 intoxication. Amino acids 1491-1971 of V. vulnificus Calcineurin governs stress survival, sexual differentiation, and virulence of 29307 RtxA1 toxin exhibits approximately 25% identity with ezrin, radixin, the human fungal pathogen Cryptococcus neoformans. Herein, we moesin (ERM) family protein sequences that function as linkers between identified and characterized calcineurin substrates in C. neoformans by the plasma membrane and actin cytoskeleton. HeLa cells expressing RtxA1 employing phosphoproteomic TiO2 enrichment and quantitative mass amino acids 1491-1971 fused to green fluorescence protein (GFP-RtxA1ERM) spectrometry. The identified targets include the zinc finger transcription became rounded and the fusion protein colocalized with actin. Through a factor Crz1 and proteins whose functions are linked to P-bodies/stress yeast two-hybrid screening and subsequent validation by immunoprecipitation granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and assay, we confirmed a specific binding of RtxA1 with host cell filamin A, an Puf4. We show that Crz1 is a bona fide calcineurin substrate, and actin-crosslinking scaffold protein. Interestingly, HeLa cells intoxicated localization and transcriptional activity of Crz1 are controlled by with RtxA1 appeared to selectively dump out cytoskeleton proteins, such calcineurin. Several of the calcineurin targets localized to PBs/SGs, as filamin, actin, and tubulin, to the culture supernatant. Downregulation including Puf4 and Pbp1, and are required for survival at high temperature of filamin A by siRNAs decreased the cytotoxicity of RtxA1 to HeLa cells. and for virulence. Genetic epistasis analysis revealed that Crz1 and the Pretreatment with a filamin A-specific antibody decreased the cytotoxicity novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin of V. vulnificus RtxA1. Phosphorylation of JNK and p38 mitogen-activated pathway that orchestrates stress survival and virulence. These findings protein kinases was located below the RtxA1-filamin A binding during the propose that calcineurin controls thermal stress and virulence at the RtxA1-mediated death of HeLa cells. These results suggest that remodeling transcriptional level via Crz1 and post-transcriptionally by regulating of cytoskeleton after filamin A interaction with RtxA1 toxin would be an target factors involved in mRNA metabolism. important cellular reaction during the programmed necrotic death of RtxA1 intoxicated cells.

46 | www.msk.or.kr Poster

E001 E003 Purification, Crystallization, and X-ray Crystallographic Ornithine Lipid-mediated Regulation of Virulence and Studies on Bacillus stearothermophilus Molybdenum Biofilm Formation in Pseudomonas aeruginosa Cofactor-dependent YiiM Soo-Kyung Kim1,2,3, Xi-Hui Li1,2,3, and Joone-Hee Lee1,2,3* Byeol Nam-gung and Sung-il Yoon* 1Lab of Microbiology, 2College of Pharmacy, 3Department of Pharmacy, Pusan Department of Biomedical Convergence, College of Biomedical Science, Kangwon National University National University Ornithine lipids (OLs) are bacteria-specific lipids that are widely found in YiiM is found in a variety of bacteria and plays a major role in the removal outer membrane of many Gram (-) bacteria, but not detected in Eukarya of toxic N-hydroxylated base analogues. YiiM converts mutatgenic 6-N- and Archaea. Pseudomonas aeruginosa produces OL under phosphate-limiting hydroxylaminopurine (HAP) to adenine in a molybdenum cofactor (MoCo) conditions and has olsBA operon encoding acyltransferases that functions dependent manner. YiiM belongs to the MoCo sulphurase C-terminal the OL biosynthesis. This olsBA operon is highly expressed under phosphate- domain (MOSC) protein superfamily. The cofactor-binding mode and limiting condition. OlsB has similar structure with LasI, an acyl-homoserine enzymatic mechanism of the MOSC family remain to be revealed. As a first lactone synthase. We addressed how OLs modulate the biofilm formation step to define the biochemical activity and structural features of YiiM, we and virulence of P. aeruginosa during the infection to host cells. The have performed expression, purification and X-ray crystallographic studies virulence of P. aeruginosa was investigated by using two host models. The on Bacillus stearothermophilus YiiM (bsYiiM). bsYiiM was overexpressed overproduction of OLs alleviated the virulence of P. aeruginosa by reducing in an Escherichia coli expression system and purified to homogeneity by the quorum sensing activity. We suggest that the olsBA overexpression chromatographic methods. bsYiiM crystals were obtained in PEG conditions sequesters the cellular acyl-group pool toward the OL synthesis from acyl and diffracted X-ray to 2.0 Å resolution. The crystals belonged to space homoserine lactone synthesis. The OL effect on biofilm formation was analyzed in flow cell system. OLs enhanced the biofilm formation of P. group P212121 with one molecule in the asymmetric unit. The bsYiiM structure was determined by molecular replacement and is currently aeruginosa. Since the hydrophobicity of cell surface increased by the OL under model building and refinement. The final structure would allow us overproduction, this may facilitate the attachment of cells to surface. The to predict the MoCo-binding site of MOSC proteins and provide their host response to OLs was investigated by measuring the expression of catalytic mechanism. 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]

E002 E004 Anthranilate Deteriorates Bacterial Biofilms Dissection of the HOG Pathway Activated by Hydrogen Peroxide in Saccharomyces cerevisiae 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 Young Mi Lee1,2, Eun Jung Kim1,2, Ji Eun An3, Ye Ji Lee4, Eun Yong Choi4, National University Won Ja Choi2,3,4, Eun Pyo Moon5, and Wan Kee Kim1* 1Department of Pharmacology, School of Medicine, Ajou University, 2Division of Anthranilate is an important intermediate for the syntheses of tryptophan Ecological Sciences, College of Natural Sciences, 3Department of Life Sciences and Pseudomonas quinolone signal in Pseudomonas spp. While some bacteria, and Division of Ecological Sciences, College of Natural Sciences, 4Interdisciplinary including P. aeruginosa, degrade tryptophan to anthranilate through a Program of EcoCreative, College of Natural Sciences, Ewha Womans University, kynurenine pathway using the kynBAU genes, other bacteria harboring 5Department of Life Sciences, College of Natural Sciences, Ajou University tnaA gene encoding tryptophanase that converts tryptophan into indole, pyruvate, and ammonia produce indole from tryptophan degradation (for Cells usually cope with oxidative stress by activating signal transduction examples, Escherichia coli, Vibrio vulnificus). Therefore, bacterial community pathways. In the budding yeast Sacchromyces cerevisiae, the high osmolarity that exists in tryptophan-rich environments contains anthranilate or indole glycerol (HOG) pathway has been implicated in transducing the oxidative (or both) necessarily. It has been recently reported that anthranilate stress-induced signal, but the underlying mechanisms are not well defined. deteriorates the biofilm structure by reducing intracellular c-di-GMP levels Based on phosphorylation of the mitogen-activated protein kinase (MAPK) and modulating the expression of Psl, Pel, and alginate in P. aeruginosa. Hog1, we revealed that the signal from hydrogen peroxide (H2O2) flows We investigated the anthranilate effect on biofilm formation of various through Ssk1, the response regulator of the two-component system of the bacteria and the underlying mechanism of the anthranilate effect. HOG pathway. Downstream signal transduction into the HOG MAPK cascade Static and flow-cell systems were used for biofilm formation and direct requires the MAP kinase kinase kinase (MAPKKK) Ssk2 but not its paralog confocal microscopic observation was carried out to monitor the anthranilate Ssk2. Activation of Ssk2 occurs in both Ssk1-dependent and -independent effect on biofilm formation. We also measured bacterial motility and manners, culminating in Hog1 phosphorylation via the MAPKK Pbs2. H2O2- intracellular c-di-GMP levels. activated Hog1 is retained in the cytoplasm, but is still able to activate the Anthranilate has a significant deteriorating effect on biofilms of Vibrio cAMP- or stress-responsive elements by unknown mechanisms. This signaling vulnificus, Bacillus subtilis, and Staphylococcus aureus. And anthranilate pathway may be used to identify oxidative stress-related MAPKKKs of significantly enhanced swimming and swarming motility of V. vulnificus other species including mammals based on the ability to complement the and B. subtilis. SSK2 deletion mutant and subsequently activate Hog1 in response to [Supported by grants from KRF] H2O2.

www.msk.or.kr | 47 2016 International Meeting of the Microbiological Society of Korea

E005 E007 Overexpression of OLE1 Enhances Stress Tolerance and Structural Analysis of Ligand Bound Complex of UdgX: A Constitutively Activates the MAPK Hog1 through the Unique Uracil DNA Glycosylase Superfamily Binding Uracil DNA MAPKKK Ssk2 Woo-Chan Ahn1,2, Min-Ho Lee1, Pau Biak Sang3, 3 1,4 Ye Ji Lee1, Olviyani Nasutution2, Young Mi Lee3, Eun Jung Kim3, Umesh Varshney , and Eui-Jeon Woo * Wan Kee Kim3, and Won Ja Choi1* 1Korea Research Institute of Bioscience and Biotechnology, 2Department of 1 2 Biological Sciences, KAIST Institute for the Biocentury, Korea Advanced Institute Interdisciplinary Program of EcoCreative, Division of Life and Pharmaceutical 3 3 of Science and Technology, Department of Microbiology & Cell Biology, IISc, Sciences, Ewha Womans University, Department of Pharmacology, School of 4 Medicine, Ajou University Bangalore, India, University of Science and Technology

Previously, it was found that the mRNA level of OLE1 increases significantly Uracil DNA glycosylases recognize and excise Uracil from DNA as the first in Saccharomyces cerevisiae cells exposed to acetic acid. Compared with step of base excision repair system, an important pathway of DNA repair control, an OLE1-overexpressing strain displayed better proton efflux, system. UdgX, the most recently discovered UDG from Mycobacterium lower membrane permeability and lessened internal hydrogen peroxide smegmatis, specifically recognizes uracil in DNA. It tightly binds to uracil in content, and enhanced tolerance to various stresses. The enhanced stress DNA. The by binding that is even stable in higly reducing conditions in SDS tolerance was considerably diminished when OLE1 was overexpressed in a gel and heat treatment with a signature loop, KRRIH. In previous biochemical strain deleted for HOG1, which encodes the mitogen-activated protein report (Sang et al (2015) 43(17) Nucl. Acids Res.) mutation of the unique kinase (MAPK) Hog1 of the high osmolarity glycerol (HOG) pathway, demonstrating loop, KRRIH, leads UdgX to cleave uracil from DNA as other Udg does. Here, its Hog1 dependency. We further found that OLE1 overexpression constitutively we report the crystal structure of UdgX, UdgX capturing uracil, UdgX H109S activates Hog1, which remains in the cytoplasm. This Hog1 activation was mutant, and complex with UdgX and DNA containing uracil. Considering accomplished through the MAPK kinase kinase (MAPKKK) Ssk2, but not this enzyme belongs to Udg family4, overall structure and catalytic residues through Ste11 and Ssk22, the other MAPKKKs of the HOG pathway. of UdgX are well conserved. The structure with uracil in active site pocket Despite its cytoplasmic location, activated Hog1 was able to activate the also verifies the conservation of catalytic motif among Udgs. In structure expression of its canonical targets such as CTT1, HSP12, and STL1, and of the complex of UdgX and DNA with uracil, however, presents unique further the cAMP response element and stress response element present features which has not reported before. The structure of deoxyuridine in in the promoter. On the other hand, OLE1 overexpression neither caused UdgX-DNA complex shows unstable conformational strain, which imply nor relieved the endoplasmic reticulum stress. Individually or in combination, the catalytic mechanism of Udgs. The unique loop possessing His109 the physiological and molecular changes caused by OLE1 overexpression locates very close to 3’ phosphate of deoxyuridine, which suggests the may contribute at least in part to enhanced tolerance to various types of stress. ground for tight binding of UdgX and DNA possessing uracil. [Supported by BK21 plus]

E006 E008 Structural and Functional Studies on Uracil DNA Glycosylase Elucidation of Regulatory Genes for Enhancement of from Bradyrhizobium japonicum Rapamycin Production

1,2 3 3 4 Vinod Vikas Patil , Ullas Valiya Chembazhi , Biak Sang Pau , Ravi Tiwari , Jin A Jung, Eun ji Kim, Jae-yeon Hwang, Myoun Su Kim, Shi Ying Jin, 3 1,2 Umesh Varshney , and Eui-Jeon Woo * Na Ryeong Lee, and Yeo Joon Yoon* 1 2 Korea Research Institute of Bioscience and Biotechnology, University of Science Department of Chemistry and Nano Science, Ewha Womans University and Technology, 3Department of Microbiology & Cell Biology, IISc, Bangalore, 4 India, School of Veterinary and Life Sciences, Murdoch University, Western Rapamycin is a 31-membered polyketide macrolide produced by Streptomyces Australia rapamycinicus. It possesses various biological and pharmacological activities including antifungal, immunosuppressive, and anti-aging activities. However, Stress conditions during plant rhizobia interactions induces DNA damage the regulatory mechanism of the pathway-specific regulators for rapamycin in rhizobia. The deamination of cytosine to uracil in DNA is one of the biosynthesis has only been partially revealed. Sequence analysis of the common cause for DNA damage that affect overall fitness of bacteria. rapamycin biosynthetic gene cluster showed that three putative negative Uracil DNA Glycosylase is an enzyme that repair DNA by base excision regulatory genes rapY, rapS, and rapR indicated high sequence similarity pathway. Here we report a novel protein Q89XRO_BRAJA (BjUng) isolated to the known TetR family proteins, response regulator, and histidine kinases, from Bradyrhizobium japonicum, an organism in which no classical homologue respectively. Overexpression of each three genes significantly reduced of family-1 UDG have been identified. Kinetic and biochemical characterization rapamycin production, while deletion of rapY and rapS induced increased of BjUng reveals it as a single strand specific UDG, which is also active on level of rapamycin production by 4.6-fold and 3.7-fold, respectively, 5-hydroxymethyl uracil and xanthine. Unlike family 1 UDGs, BjUng is compared to that of the wild-type strain. Gene expression analysis by impervious to inhibition by Ugi, a phage protein which specifically inhibits RT-PCR showed that rapS negatively controlled the expression of rapY and the family 1 UDGs, and the abasic DNA (AP-DNA). BjUng homologues are rapX encoding a putative ABC-transporter was negatively controlled by present in many other microbes and our phylogenetic analysis reveals that rapY. Interestingly, overexpression of rapX in the rapS deletion mutant this protein constitutes a previously unidentified family of UDGs. Crystal accomplished an increase in rapamycin production compared to the structure of BjUng shows striking similarity with the family 1 UDGs in wild-type strain. These results demonstrate the roles of rapY, rapR, and terms of key active site residues and overall fold, suggesting its origins rapS as negative regulators of rapamycin biosynthesis in S. rapamycinicus through divergence. Bjung exist as a dimer with seven α-helix and six β- and shed light on understanding the complex regulatory mechanism and sheets. One ligand binding site is present per monomer of Bjung. Asp57, increasing the level of rapamycin production in industrial strains. Glu62, Leu69, Asn94, His168 are critical residues involved in uracil binding. [Supported by NRF, ICT and MISP.] Our studies on Bjung shed light on structural and functional aspect of DNA repair system in rizobacteria.

48 | www.msk.or.kr Poster

E009 E011 Localization of an Acid Responsive Element in the Laccase Trans-4-hydroxy-l-proline: A Novel Constituent of Promotor Expressed at Low pH in congregatus Compatible Solute in Moderate Halophilic Bacteria

Su Yeon Kim1, Linh Trieu Dieu Nguyen1,2, and Hyung Tae Choi1* Kyung Hyun Kim, Baolei Jia, and Che Ok Jeon* 1Molecular Microbiology Lab, Department of Biochemistry, Kangwon National Department of Life Science, Chung-Ang University University, 2Vietnam It has been reported that Halobacillus halophilus, a moderate halophilic Coprinellus congregates, an inky cap, synthesizes and secretes laccase at bacterium, having a capability to grow a wide range of salinities (0.5–3.0 M acidic pH (pH 4.0-4.5) transiently only in the dikaryotic mycelium. Different NaCl) has a strategy to accumulate osmolytes such as glutamate, glutamine, lengths of expression vectors were constructed using the vector pBARGEM7-1 and proline to cope with osmotic stress under high salt environments. The inserted with green fluorescent protein (GFP) reporter gene regulated by analysis of organic compounds using 1H-NMR in H. halophilus showed that different lengths of the laccase promoter (F0-F5) obtained from 5’-sequential trans-4-hydroxy-l-proline (Hyp), which has not been reported as a compatible deletion constructs, and they were introduced to wild type strains (a1 & solute in previous studies, increased depending on NaCl concentrations, a2) to determine the dikaryon-responsive and acid-stress responsive DNA suggesting that Hyp might be an important compatible solute in H. elements. The transformants were confirmed by PCR with the specific halophilus. The genome data in GenBank showed that H. halophilus does primers which were used in cloning. Monokaryotic transformants of not harbor a proline 4-hydroxylase (P4H) gene, responsible for the conversion C.congregatus monokaryons were used to generate dikaryons. These from proline to Hyp. Based on BLAST and Pfam analysis using genes annotated transformants were grown in neutral pH liquid media, and then transferred as P4H in other bacteria, a putative P4H gene, which was annotated as a to acidic media to examine the GFP expression by Confocal Microscope. multidrug DMT transporter permease was selected and its expressional The homozygotic transformants which had full-length (2.0 kb promoter) enzyme in E.coli strain EL21 showed a clear converting activity from proline showed the GFP expression under acidic condition, while the shortest- to Hyp. Transcriptional analysis also showed that the expression of the length-promoter did not show GFP expression. We derived a conclusion putative P4H gene increased at high NaCl conditions. In addition, Hyp was that acid stress element localize in the various promotor region between detected from other moderate halophilic bacteria. These results suggest cloned length (F0-F5). We have also constructed shorter expression vector that Hyp may be a ubiquitous compatible solute in moderate halophilic which had shorter lengths of promoter (F6, F7) to confirm this experiment. bacteria. [Supported by grants from University-Industry Cooperation Foundation] [This work was supported by the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ00999302)”, RDA, Republic of Korea.]

E010 E012 The Sugar-dependent Regulation of C-di-GMP and Signal Development of a Colorimetric Quantification Method for Transduction System Characterization of Lactic Acid Bacteria

Kyoo Heo1, Young-Ha Park1, and Yeong-Jae Seok1,2* Min Young Jung, Sung-Oh Sohn, Se Hee Lee, Boyeon Park, 1Department of Biological Sciences and Institute of Microbiology, 2Department Hae Woong Park, and Jong-Hee Lee* of Biophysics and Chemical Biology, Seoul National University World Institute of Kimchi

Vibrio cholerae, the causative pathogen of severe diarrheal disease, inhabits Rapid colorimetric methods using various indicator reagents have been various niches, including human host. Previous studies uncovered that V. developed to monitor bacterial viability. Here, we examined the applicability cholerae undergoes adaptation to these environments, which alter their of a method based on the reduction of resazurin or water-soluble tetrazolium behaviors and various virulence gene expressions. However, the exact salt-8 (WST-8) to screen lactic acid bacteria (LAB) for tolerance against bile mechanism regulating these adaptations has yet to be found. Recent salts and low pH. The resazurin reduction test proved unsuitable for studies have suggested that c-di-GMP, a common secondary messenger screening LAB such as Lactobacillus plantarum and Leuconostoc mesenteroides found in most bacteria, is involved in the regulation of the virulence of since it reacted with lactic acid present in the cultures. LAB growth could many pathogens, including V. cholerae, which is dependent on various be indirectly quantified by measuring WST-8 reduction. This method factors such as biofilm formation, motility and toxin production. There are proved more sensitive than measuring bacterial colony forming units and more than 50 proteins involved in controlling the concentration of c-di-GMP turbidity at 600 nm. Our results indicate that the WST-8-based method in Vibrio cholerae. Diguanylate cyclases which have a GGDEF domain synthesize can be used to identify probiotic characteristics in LAB. 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]

www.msk.or.kr | 49 2016 International Meeting of the Microbiological Society of Korea

E013 E015 Investigation of the Interaction between HPr and FruR in OxyR-dependent Gene Expression Involved in Exopolysaccharide Vibrio cholera Production in Acinetobacter oleivorans DR1

Chang-Kyu Yoon1, Hey-Min Kim1, Young-Ha Park1, Bora Shin and Woojun Park* 1 1,2 Yeon-Ran Kim , and Yeong-Jae Seok * Department of Environmental Science and Ecological Engineering, Korea university 1Department of Biological Sciences and Institute of Microbiology, 2Department of Biophysics and Chemical Biology, Seoul National University Many bacteria secrete exopolysaccharides (EPS) that contribute to their nutrient trapping, surface attachment, and protection against abiotic or Vibrio cholera is known as a human opportunistic pathogen which infects biotic stresses. Like other Acinetobacter species the genome of Acinetobacter hosts through contaminated water and food. Many bacterial species including oleivorans DR1 contains three distinct EPS operons [two PNAG (poly-N-acetyl V. cholera possess the phosphotransferase system (PTS) as a sugar transport glucosamine) operons and the K locus]. Our quantitative RT-PCR analysis system. The sugar PTS consists of sugar-specific enzyme II proteins and showed that the levels of their expression vary under different EPS-producing two general components, enzyme I and HPr. These PTS components play conditions: H2O2, hexadecane, NaCl, and cold temperature. Morphologically critical roles in transport of sugars from outside of cells by sequential distinct biofilms by those abiotic stresses were observed using confocal phosphoryl transfer. In addition to sugar uptake and phosphorylation, the laser scanning microscopy, indicating involvement of different cellular components sugar PTS regulates other functions through protein-protein interaction in in each biofilm formation. Promoter and expression analyses of EPS genes a phosphorylation state-dependent manner. Although roles of the PTS indicated involvement of the H2O2-sensing OxyR regulator in controlling have been extensively studied in Escherichia coli, physiological roles of PTS three EPS operons. Interestingly, OxyR displayed different degree of binding are barely known in V. cholera. In this study, FruR has been found as a new to the promoters of each EPS operon. Biofilm formation was slightly protein partner interacting with HPr in V. cholera. FruR is known as a inhibited in the oxyR mutant compared to the wild-type strain under abiotic GalR/LacI superfamily of transcriptional regulator which regulates expression stress conditions. However, total EPS amounts in the mutant were not of the genes related in the metabolism and transport of fructose through changed, which suggested that other factors could contribute to production the direct binding to the cognate operator sequence of the operon of of different EPS. Further investigation is needed for understanding regulation fructose PTS. Only the dephosphorylated form of HPr interacts with FruR of OxyR-dependent and -independent EPS productions and their contribution and the HPr-FruR complex can bind to DNA fragment containing the to biofilm formation. putative FruR binding sequence. It is assumed that HPr has an effect on [This work was supported by grants from the National Research Foundation the FruR-dependent regulation as a co-repressor in the presence of of Korea (NRF).] glucose. [This work was supported by the Samsung Science & Technology Foundation and National Research Foundation (NRF).]

E014 E016 Generation of a FK506 Analogue through Genetic The Structural Insights of a Cytosol Protein Disulfide Engineering of Streptomyces Strain Reductase DsbM from Pseudomonas aeruginosa

Xu Zhao, Hea Luying Shin, Heqing Cui, Ji Yoon Beom, Inseong Jo1, In-Young Chung2, You-Hee Cho2, and Nam-Chul Ha1* Ji Young Lee, and Yeo Joon Yoon* 1Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Department of Chemistry and Nano Science, Ewha Womans University Research Institute for Agricultural and Life Sciences, Seoul National University 2Department of Pharmacy, College of Pharmacy, CHA University FK506 is a clinically used immunosuppressant originally isolated from Streptomyces tsukubaensis. In addition to its immunosuppressive action, In bacteria, many Dsb family proteins play diverse roles in the conversion FK506 has been reported to exhibit antifungal, anti-inflammatory, neuroprotective, between the oxidized and the reduced states of cysteine residues of and neuroregenerative activities. FK506 is synthesized by a hybrid polyketide substrate proteins. Most Dsb family proteins target proteins in the synthase/nonribosomal peptide synthetase (PKS/NRPS) system. Through periplasmic or extracellular regions of bacteria. Recently, DsbM proteins our efforts characterizing the functional role of enzymes FkbM and FkbD were found in some Gram-negative bacteria. It is a cytosolic Dsb member and identifying the biosynthetic intermediates present in the two parallel with the conserved CXXC motif, and it was implicated in reducing of the pathways for the post-PKS modification of FK506 biosynthesis, a new cytoplasmic redox sensor protein OxyR in Pseudomonas aeruginosa. Here, FK506 analogue (9-deoxo-prolyl-FK506) was isolated and identified from we present the crystal structures of DsbM from P. aeruginosa, revealing the FkbD deletion mutant of Streptomyces sp. KCTC11604BP. Additionally, that it consists of a modified thioredoxin domain containing the CXXC 9-deoxo-prolyl-FK506 exhibited reduced in vitro immunosuppressive activity motif and a lid domain surrounding the CXXC motif. In a glutathione-linked but similar neuroregenerative activity in comparison to FK506. This study structure, a glutathione molecule is linked to the CXXC motif of DsbM and demonstrates the successful application of biosynthetic engineering approaches is bound in an elongated canyon-like region in the thioredoxin domain, including the deletion of a biosynthetic gene from a gene cluster. In which is also suited for substrate peptide binding. A striking structural conclusion, this research demonstrates that neuroregenerative drugs can similarity to a human glutathione S-transferase was found as the glutathione be generated from the FK506 macrolide class of natural products without binding pocket was shared. We further presented biochemical evidence the side effects attributed to immunosuppression. 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.

50 | www.msk.or.kr Poster

E017 E019 Cellular Responses of Hemolytic Bacillus cereus MH-2 Physiological Activities of Two Wine Yeasts, Pichia Species Exposed to Epigallocatechin Gallate (EGCG) Isolated from Crushed Grapes

Dong-Min Kim, Sang-Kook Park, and Kye-Heon Oh* Sang-Kook Park, Dong-Min Kim, and Kye-Heon Oh* Department of Life Science and Technology, Soonchunhyang University Department of Life Science and Technology, Soonchunhyang University

The aim of this work was to investigate the hemolytic activity and The aim of this work was to examine the characteristics and functional characterization of Bacillus cereus MH-2 exposed to EGCG, which produces activities of wine yeast Pichia species. Initially, two yeasts were isolated the hemolytic activity isolated from commercial Ssam-Jang. Initially, the from crushed grapes. Using 16S rRNA sequencing, two strains could be physiological and biochemical characteristics of strain MH-2 was obtained assigned to Pichia manshurica and Pichia terricola. Physiological and from the Biolog system. Analysis of the carbohydrate utilization profiles biochemical characteristics of strain MH-2 was examined. During the based on the GP2 MicroPlate and 16S rRNA sequence also placed MH-2 as incubation period, several physiological activities (e.g., angiotensin-converting a Bacillus cereus with a confidence of 100%, and the strain was designated enzyme inhibition, tyrosinase inhibitory activity, SOD-like activity, DPPH as B. cereus MH-2, and registered as [KU885976]. Survival of the MH-2 inhibition) of two strains were evaluated and compared. Based on the strain with time in the presence of different concentrations of EGCG under results, two wine yeast, P. manshurica and P. terricola might be included sublethal conditions was monitored, and viable counts paralleled the as probiotics, presenting the potential to be used in the functional foods production of the stress shock proteins in this bacterium. Analysis of for human as well as animal health. 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 E020 Isolation and Cellular Responses of Explosive Karyopherins Involved in the Nuclear Actin Transport HMX-degrading Bacterium and Its Morphological Changes Under Sublethal HMX Concentrations Immanuel Dhanasingh and Sung Haeng Lee* Department of Cellular and Molecular Medicine, Chosun University School of Dong-Min Kim, Sang-Kook Park, and Kye-Heon Oh* Medicine

Department of Life Science and Technology, Soonchunhyang University Although compartmentalization of a eukaryotic cell into the nucleus and cytoplasm has benefits, it further necessitates transport between the two Soil samples were collected from explosive manufacturing plant sites and compartments. Active transport between these two compartments requires used for enrichment of microbial consortia with HMX (1,3,5,7-tetranitro- metabolic energy and transporter proteins. Nuclear transporter receptors 1,3,5,7-tetrazocane) under aerobic and nitrogen-limiting conditions. Among belong to the karyopherin super family and aid in the movement of cargo five isolates from microbial consortia using HMX as substrate for growth, into (importins) or out of the nucleus (exportins) or are bidirectional. an isolate, HK-5 which has excellent HMX degradability was selected for Presence of actin in nucleus and its nuclear trafficking remains intriguing this work. Both BIOLOG system and 16S rRNA sequencing were conducted due to its lack of nuclear import or export signals. Despite the fact that to identify the strain, which was assigned to Pseudomonas plecoglossida importin-9 and exportin-6 have been linked to nuclear import and export HK-5. Complete degradation of 75 uM HMX was achieved after 50 days of of actin respectively, the mechanistic details about actin-karyopherin incubation. Analysis of SDS-PAGE and Western blot revealed that stress interaction are not known yet. We compared the structural details of shock proteins, DnaK and GroEL, specifically reacting with anti-DnaK and already available karyopherins and provide the initial structural prediction anti-GroEL monoclonal antibodies were produced when cells were for exportin-6. The WH2 domain from diverse actin binding proteins treated with HMX. Scanning electron microscopic analysis demonstrated display a similar architecture of N-terminal α helix followed by the LKKT(V) the presence of cells with wrinkled surfaces containing perforations and motif. A similar LKPS motif was identified near helix 14A of exportin-6, irregular rod-shaped forms after exposure to HMX. which might be the binding site for actin. Based on these predictions, we have postulated a mechanism of actin-karyopherin interaction.

www.msk.or.kr | 51 2016 International Meeting of the Microbiological Society of Korea

E021 E023 Crystal Structure of Glycogen Branching Enzyme from Bacillus licheniformis Contains Two More PerR-like Proteins Pyrococcus horikoshii in Addition to PerR, Fur and Zur Orthologues

Soo Hui Na and Nam Chul Ha* Yoon Mo Yang, Jung Hoon Kim, Su Hyun Ryu, Yeh Eun Lee, and Jin Won Lee* Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Department of Life Science and Research Institute for Natural Sciences, Hanyang Seoul National University University

Glycogen branching enzyme (GBE) is a branching enzyme that catalyzes The ferric uptake regulator (Fur) family proteins include sensors of Fe the formation of α1,6-branching points during glycogenesis by cleaving α (Fur), Zn (Zur), and peroxide (PerR). Among Fur family proteins, Fur and 1,4 bond and making a new α1,6 bond. Here, we determine the crystal Zur are ubiquitous in most prokaryotic organisms, whereas PerR exists structure of GBE from the hyperthermophilic archaea, Pyrococcus horikoshii, mainly in Gram positive bacteria as a functional homologue of OxyR. Gram and PhGBE belongs to glycoside hydrolase 57 (GH57). The crystal of GBE positive bacteria such as Bacillus subtilis, Listeria monocytogenes and belongs to P21 space group with unit cell dimensions of a = 121.517 Å, b = Staphylococcus aureus encode three Fur family proteins: Fur, Zur and 43.021 Å and c = 122.682 Å. The final structure of GBE was refined at a PerR. In this study, we identified five Fur family proteins from B. licheniformis: resolution of 2.0 Å with and R factor of 18.7% and an R free of 24.7%. The two novel PerR-like proteins (BL00690 and BL00950) in addition to Fur enzyme has a central (β/α)7 fold catalytic domain and an α-helix-rich (BL05249), Zur (BL03703), and PerR (BL00075) homologues. Our data indicate C-terminal domain, which participates in the formation of the active-site that all of the five B. licheniformis Fur homologues contain a structural Zn2+ cleft. We found vanillyl acetone as a activator candidate for this enzyme in site composed of four cysteine residues like many other Fur family proteins. Natural Compound Library. 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].]

E022 E024 Crystal Structure of the Regulatory Domain of AphB, a Effect of Exogenous Glutamine on Salmonella Replication Virulence Gene Activator from Vibrio vulnificus under Nitrosative Stress Conditions

Nohra Park, Saemee Song, Inseong Jo, Sang Ho Choi*, and Nam-Chul Ha* Yoon Mee Park and Iel Soo Bang* Department of Agricultural Biotechnology, College of Agriculture and Life Department of Microbiology and Immunology, Chosun University School of Sciences, Seoul National University Dentistry

Many pathogenic bacteria increases expression of virulence factors by Nitric oxide (NO) damages various bacterial macromolecules, resulting in recognizing the host environments.Transcriptional activator AphB has been abnormal metabolism and subsequent growth arrest. But, the NO targets known as a thiol-based switches to sense the anoxic host environment and strategies for avoiding nitrosative stress in bacteria are still not largely and turn on the transcription of virulence genes in V. vulnificus and Vibrio understood In previous studies, we have shown that NO can cause auxotrophy cholerae. Crystal structures of the full-length AphB from V. cholerae was for amino acids in hmp mutant S. Typhimurium that can hardly metabolize determined, reporting the tetrameric assembly of AphB whose protomer NO entering into bacteria. This study presents that exogenous supplementation adopts the typical structures of the LysR-type family proteins consisting of with glutamine (Gln) or glutamate (Glu) can fully restore the replication of the DNA binding domain and the regulatory domain (RD). The conserved hmp mutant S. Typhimurium in NO-producing cultures. Because reactions cysteine residue in RD has implicated that it senses the oxygen very for the biosynthesis of Gln and Glu are interrelated, we analyzed the effect sensitively and plays a key role in keeping the structure of AphB at the of supplementation with Gln or Glu on the growth of hmp mutants further inactive state. In this study, we determine the crystal structure of AphB RD lacking genes for the biosynthesis of Gln and/or Glu, and found that, in the at 1.9 Å resolution under aerobic conditions. The structure reveals that presence of NO, Salmonella required Gln for their growth. Only L-form of the conserved cysteine residue is buried in the interior region and does Gln could restore bacterial growth, suggesting that this growth recovery not undergo any oxidation or modification under the aerobic condition. may have less relation with bacterial cell wall biosynthesis. The Gln Ensuing biochemical studies also shows that the cysteine residue barely supplementation did not affect the NO consumption rate of wild type and react with the environmental stresses such as hydrogen peroxide, nitric hmp mutant S. Typhimurium, indicating that the Gln-promoted NO resistance is oxide, and alkyl hydrogenperoxide. Our findings suggests that the cysteine independent of NO metabolism itself. These results suggest that cytotoxic residue might not be important in sensing the environmental changes NO may impair reactions for maintaining levels of Gln in S. Typhimurium, during the pathogenesis. Further studies are required to elucidate the resulting in auxotrophy for Gln, but which can be overcome by bacterial molecular mechanism. taking up Gln from surroundings. [Supported by NRF grant (2008-0062283)]

52 | www.msk.or.kr Poster

E025 E026 Crystal Structure of Bacterial 1-Cys Peroxiredoxin from The HPr-pyruvate Kinase Complex Protects Vibrio vulnificus

Vibrio vulnificus and Its Structural and Functional Cells against H2O2 Stress Generated by Fungal Neighbors in Implications to Scavenging ROS and Nitric Oxide the Presence of Glucose

1 1 1 2 Jinsook Ahn , Kyung Ku Jang , Inseong Jo , Jin-Wook Yoo , Hey-Min Kim1, Young-Ha Park1, Chang-Kyu Yoon1, and Yeong-Jae Seok1,2* Sang Ho Choi1, and Nam-Chul Ha1* 1Department of Biological Sciences and Institute of Microbiology, 2Department 1 Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, of Biophysics and Chemical Biology, Seoul National University Center for Food and Bioconvergence, Research Institute for Agricultural and Life 2 Sciences, Seoul National University, Department of Manufacturing Pharmacy, We recently reported that dephospho-HPr increases the cellular level of Pusan National University pyruvate by interacting with pyruvate kinase A (PykA) in Vibrio vulnificus, but not in Escherichia coli. Here, we show that this interaction confers cell Peroxiredoxins are the ubiquitous cysteine-based peroxidase enzymes. survival under H O stress by stimulating PykA-mediated pyruvate production in Recently, a new type Peroxiredoxin, called VvPrx3, was identified in a 2 2 the presence of glucose. A pykA deletion mutant was more susceptible to pathogenic bacterium Vibrio vulnificus as an essential gene for survival in H O than wild-type V. vulnificus despite no significant difference in the macrophage. It belongs to 1-Cys peroxiredoxin family that contains only one 2 2 expression level of catalase, and there was a more remarkable difference catalytic cysteine residue (Cys48). Interestingly, VvPrx3 was induced in of H O sensitivity between wild-type and pykA mutant strains in the response to nitric oxide (NO) treatment, different from other types of Prx in 2 2 presence of glucose than galactose. Because some fungi are known to V. vulnificus. Here, we determine the crystal structures of VvPrx3 from V. produce hydrogen peroxide through the reaction catalyzed by glucose vulnificus both in the reduced and the oxidized forms at high resolutions. oxidase, we tested whether fungi isolated from the natural habitat of V. The crystal structure in the reduced form showed a typical dimeric interface vulnificus exert an inhibitory effect on V. vulnificus survival. While Aspergillus (A-type contact), and the structure at the oxidized state revealed a novel fumigatus did not produce H O and thus its culture filtrate did not kill V. oligomeric interface, called C-type contact, between the typical dimer. The 2 2 vulnificus cells regardless of the sugar source, A. welwitschiae produced C-type contact of VvPrx3 is mediated by a disulfide bond between the H O and showed bactericidal activity only in the presence of glucose. The catalytic cysteine residues and induced by treatment of peroxides. Ensuing 2 2 pykA mutant showed more severe growth retardation than wild-type V. studies showed that the disulfide bond of VvPrx3 is also made by the NO vulnificus in the culture filtrate of glucose-grown A. welwitschiae and this treatment, and VvPrx3 is important in survival of the bacteria in the sensitivity was rescued by the addition of pyruvate. These data suggest presence of NO. Taken together, we propose a novel function of 1-Cys that V. vulnificus resists the killing activity of its fungal neighbors by peroxiredoxins in direct scavenging of NO stress via a novel type of increasing pyruvate production in the presence of glucose. oligomerization, and our findings would help understand diverse functions [Supported by NRF] of peroxiredoxins during pathogenic process at the molecular level. [Supported by grants from SHC and NCH]

www.msk.or.kr | 53 2016 International Meeting of the Microbiological Society of Korea

F001 F003 Complete Genome Analysis of Vibrio parahaemolyticus Metabolic Role of MADS-box Transcription Factor Mbx2 in FORC_023, Isolated from Storage Water for Raw Fish Schizosaccharomyces pombe

Han Young Chung, Byungho Lee, Eun Jung Na, and Sang Ho Choi* Youngdae Seo and Jung-Hye Roe* Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, School of Biological Sciences, College of Natural Science, Seoul National University Seoul National University In eukaryotes, glucose is mainly assimilated through the respiration pathway in Vibrio parahaemolyticus is a Gram-negative bacterium and consumption mitochondria for maximum energy yield in the presence of oxygen. of seafood contaminated with this bacterium can cause gastroenteritis. V. However, yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces parahaemolyticus FORC_023 strain was isolated from the storage water pombe undergo aerobic fermentation in which glucose is predominantly for raw fish, and its whole genome was sequenced. The genome FORC_023 fermented to ethanol even in the presence of oxygen. In this study, we is composed of two circular chromosomes without plasmid. Chromosomes examined the role of a putative transcription factor Mbx2 with MADS-box consist of 4,227 predicted open reading frames (ORFs), 131 tRNA genes, in energy metabolism. Growth of mbx2Δ in minimal media was monitored and 37 rRNA genes. Among various V. parahaemolyticus, FORC_023 shows by spectrophotometer. The doubling time and the final OD of mbx2Δ the highest average nucleotide identity (ANI) value with UCM-V493 which increased about 30% and decreased about 30%, respectively, compared is one of the most virulent strains, indicating that FORC_023 is virulent as with the wild type. The entry time point of stationary growth phase was well. Furthermore, comparative genome analysis of the two strains revealed similar between the wild type and the mutant. The amount of glucose in that FORC_023 carries an additional genomic region encoding strong virulence cell cultured media, however, was not depleted at the same time. Whereas factors such as repeats-in-toxin (RTX) and type II secretion, which could the wild type consumed almost all the glucose by the onset of stationary lead to host cell death. In addition, in vitro cytotoxicity testing suggests phase, glucose in the mutant culture media was depleted far after entering that FORC_023 exhibits a higher level of cytotoxicity toward the INT-407 the stationary phase. The glucose uptake level per cell, however, showed human epithelial cells as determined by lactose dehydrogenase (LDH) little difference between the wild type and the mutant. Measurement of release. All these results suggest that the FORC_023 isolated from storage the respiration rates throughout growth phases revealed that the oxygen water for raw fish is one of the most virulent strains of V. parahaemolyticus, consumption rate was reduced by about 30% in the mutant, compare with providing new insights into the necessity for rapid detection as well as the wild type. The mechanism behind this metabolic function is being epidemiological investigation to prevent food-borne outbreaks by V. investigated. parahaemolyticus. [This research was supported by a grant (14162MFDS172) from Ministry of Food and Drug Safety in 2015.]

F002 F004 Complete Genome Sequence of Antibiotic and Anticancer Role of Rsv1 in Responding to Glucose-starvation in Agent Violacein Producing Massilia sp. Strain NR 4-1 Schizosaccharomyces pombe

Nu Ri Myeong, Hoon Je Seong, Hye-Jin Kim, and Woo Jun Sul* Eun-Jung Kim and Jung-Hye Roe* Department of Systems Biotechnology, Chung-Ang University School of Biological Sciences, College of Natural Science, Seoul National University

Massilia sp. NR 4-1 was a violacein producing strain newly isolated from In natural environment cells are faced with various conditions that cause topsoil under nutmeg tree, Torreya nucifera in Korean national monument metabolic stresses. Metabolic stresses are caused by diverse nutrients Bijarim Forest, Jeju. Massilia sp. NR 4-1 was identified as producing the depletion, and among the nutrients glucose is critical as an energy and violet pigments (violacein and deoxyviolacein). Violacein is a novel class of carbon source. Cells can adjust their physiological states upon glucose drug exhibiting anticancer and antibiotic activities originated from L-tryptophan. shortage in transcriptional and post-translational levels. In the fission Here, we present the complete genome of Massilia sp. strain NR 4-1 of yeast Schizosaccharomyces pombe, Rsv1 protein with C2H2-type zinc 6,361,416 bp and total 5,285 coding sequences (CDSs) including a complete finger domain acts as a transcription factor to maintain cellular physiological violacein biosynthesis pathway, vioABCDE. Presence of five genes involved function in glucose-starved condition. Rsv1 protein is expressed under low in violacein production pathway was identified. The genome sequence of glucose condition, but not under glucose-depleted (no glucose) condition. Massilia sp NR 4-1 will provide stable and efficient biotechnological applications When rsv1 gene-deleted mutant cells are transferred to low glucose of violacein production. 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.

54 | www.msk.or.kr Poster

F005 F007 A Genome-wide Transcriptomic Analysis of Oxidative Stress Response of Deinococcus geothermalis via Radiation-responsive Genes in the Radiation-resistant a Cystine Importer Fungus, C. neoformans Minwook Kim and Sung-Jae Lee* 1 1 1 Kwang-Woo Jung , Min-Kyu Kim , Dongho Kim , Department of Biology, Kyung Hee University Sangyong Lim1, and Yong-Sun Bahn2* 1 Research Division for Biotechnology, Korea Atomic Energy Research Institute The responses to oxidative stress induced by H2O2 of the wild-type and a 2Department of Biotechnology, College of Life Science and Biotechnology, Yonsei dgeo_1985 gene-disrupted mutant strain of Deinococcus geothermalis University were phenotypically different. At a certain concentration of H2O2, the ∆ dgeo_1985R strain was more resistant to oxidative stress than was the The basidiomycetous fungus Cryptococcus has been known as radiation wild-type strain, as determined using bacterial growth and viability assays. resistant fungi and is found in highly radioactive environments such as the The wild-type and mutant strains expressed equal levels of superoxide damaged nuclear reactor at Chernobyl. Although Cryptococcus exhibits dismutase and catalase under H2O2-induced stress. Although the expression greater resistant for gamma radiation than model yeast Saccharomyces levels of the general DNA-damage response-related genes recA, pprA, cerevisiae, the resistant mechanism of gamma radiation remains elusive. ddrA, and ddrB were up-regulated by more five-fold in the wild-type strain To elucidate a unique regulatory system for radiation-resistance in C. relative to those in the ∆dgeo_1985R mutant strain, the mutant strain neoformans, we performed genome-wide comparative analysis through had a higher survival rate than that of the wild-type when various types of DNA microarray analysis using C. neoformans WT strain (serotype A, H99 stress were induced. A cystine-transporter gene (dgeo_1986) was highly strain) responding gamma radiation. Based on the transcriptome analysis, expressed in the ∆dgeo_1985R mutant strain, at a level that was greater genes involved in DNA damage repair systems (RAD51, RDH54, and RAD54) than 150-fold that of the wild-type strain, leading to the conclusion that were significantly increased in response to gamma radiation. Actually, this cystine transporter might be involved in the defensive response to rad54Δ and rdh54Δ mutants exhibited sensitivity against both gamma H2O2 stress. In this study, the cystine transporter was identified and radiation and DNA damage inducers. Furthermore, genes regarding to characterized through membrane protein-expression analysis, a cystine-binding molecular chaperone and ubiquitination systems were strongly induced. assay, and assays of the intracellular H2O2, cysteine, and thiol levels. In contrast, expression levels of genes related to protein synthesis, fatty Therefore, this cystine-uptake system functions as a primitive defense acids/sterols synthesis, and other cellular molecules. Especially, ergosterol system that non-enzymatically scavenges oxidative stress agents in D. homeostasis is required for gamma radiation resistance. Furthermore, geothermalis. 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 F008 Crystal Structure of Thermoplasma acidophilum XerA Complete Genome Sequence of Vibrio parahaemolyticus Recombinase Shows Large C-shape Clamp Conformation FORC_022, a Food-borne Pathogen from Soy Sauce and cis-cleavage Mode for Nucleophilic Tyrosine Marinated Crab in South Korea

Chang Hwa Jo1, Junsoo Kim1, Ah-reum Han1, Sam Yong Park2, Byungho Lee, Han Young Chung, Suyeon Kim, Eun Jung Na, and Sang Ho Choi* 1 3 Kwang Yeon Hwang , and Ki Hyun Nam * Foodborne-pathogens Omics Research Center, Department of Food Science and 1Division of Biotechnology, College of Life Sciences & Biotechnology, Korea Biotechnology, Seoul National University University, 2Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Japan, 3Pohang Accelerator Laboratory, Pohang Vibrio parahaemolyticus is a curved rod-shaped bacterium that causes University of Science and Technology gastrointestinal illness associated with the consumption of seafood. To characterize V. parahaemolyticus that have caused outbreaks in South The Xer recombinase, a site-specific tyrosine recombinase, plays an important Korea, ten strains of V. parahaemolyticus were obtained from Korean role in a variety of biological processes, including excisional recombination Ministry of Food and Drug Safety. Virulence gene-specific PCR screening of plasmid DNA, and resolution of catenated DNA circles and host chromosome and lactate dehydrogenase (LDH) release assay were employed to evaluate DNA. Xer monomers bind to two double-stranded DNA dif sites and mediate the virulence of these strains. Among strains, the H8 strain, which was breaking, exchange, and rejoining of four DNA phosphodiester bonds. isolated from a crab marinated with soy sauce, had the many virulence Here we report the 2.5 Å crystal structure of XerA from the archaean genes and the strain showed a high level of cytotoxicity in the LDH release Thermoplasma acidophilum. Crystallographic data reveal a uniquely open assay. Therefore, the H8 strain was selected for genome sequencing and conformational state, resulting in a C-shaped clamp with an angle of ~48° was designated as FORC_022. The FORC_022 genome consists of two and a distance of 57 Å between the core-binding and the catalytic chromosomes and a plasmid, which have the GC content of 45.2%, domains. The catalytic nucleophile, Tyr264, is positioned in cis-cleavage 45.37%, and 44.5%, respectively. The genome was predicted to have 4,858 mode by XerA’s C-term tail that interacts with the CAT domain of a open reading frames, 133 tRNA genes, and 37 rRNA genes. Average nucleotide neighboring monomer without DNA substrate. Structural comparisons of identity analysis using nine complete V. parahaemolyticus genome sequences tyrosine recombinases elucidate the dynamics of Xer recombinase. revealed that FORC_022 was closely related to clinical strains. Furthermore, [This work was supported by the National Research Foundation of Korea virulence factors of FORC_022 were identified using BLAST against Virulence (NRF) grant funded by the Korea government (MEST) (NRF-2014R1A1A2059440 Factor Database. This study will deepen our understanding of the pathogenesis and 2013R1A1A2008404)] 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.]

www.msk.or.kr | 55 2016 International Meeting of the Microbiological Society of Korea

F009 F011 Transcriptome Analyses of a Novel Transcriptional Role of Conserved Residues within the Wedge Domain of Regulator HpxA Essential for Hypoxic Responses in Deinococcus radiodurans RecG Aspergillus nidulans Using RNA-Seq Sun Wook Jeong, Jing Zhang, Lei Zhao, Min Kyu Kim, and Sangyong Lim* Sun-Ki Koh, Dawoon Chung, Jun-Yong Kwak, Research Division Biotechnology, Korea Atomic Energy Research Institute Mee-Hyang Jeon, and Suhn-Kee Chae* Department of Biochemistry, Paichai University Deinococcus radiodurans has exceptional ability to repair DNA damage caused by various DNA-damaging agents including ionizing radiation (IR). Hypoxia-sensitive hpxA mutant alleles and ΔhpxA in Aspergillus nidulans Some deinococcal Rec proteins are known to be different from counterparts of the IR-sensitive bacterium Escherichia coli in structure and function. showed completely blocked growth in hypoxia (1% O2) and significantly increased susceptibility to an antifungal drug itraconazole compared to RecG helicase is highly evolutionarily conserved in bacteria and required wild type (WT). These phenotypes were consistent with the null-mutant for efficient DNA repair. The comparison of amino acid sequence revealed phenotypes of srbA encoding a sterol regulatory element binding protein that three amino acids ‘201QPW203’ located in the wedge domain responsible (SREBP), a transcriptional regulator critical for hypoxia adaptation in fungi. for DNA binding are well conserved in Deinococcus RecG. We constructed To investigate genes whose transcriptions are regulated by HpxA, we the DrRecG derivative DrRecGFSA, in which ‘QPW’ was substituted by the performed RNA sequencing analyses with ΔhpxA and WT strains under equivalent of ‘99FSA101’ in EcRecG, and the EcRecG derivative EcRecGQPW, hypoxic conditions. A total of 183 genes were down-regulated in ΔhpxA by in which ‘FSA’ was substituted by ‘QPW’. EcRecGQPW partially complemented Δ more than 4-fold relative to WT, while 300 genes were up-regulated in ΔhpxA. recG in trans under MMC-stress conditions, whereas DrRecGFSA did not Δ Interestingly, srbA transcription levels were decreased in ΔhpxA compared complement recGDR at all like EcRecG. DNA binding activities of the to WT, and several SrbA regulons involved in oxygen-consuming processes purified recombinant wedge domains of DrRecG and EcRecGQPW were ∆ including syntheses of ergosterol, heme, and coenzyme Q10 were also higher than that of EcRecG. We further examined DNA repair kinetics in identified as HpxA target genes. To elucidate a link between HpxA and recGDR containing RecG derivatives and found that the genome shattered ∆ SrbA, we conducted quantitative real-time PCR with ΔhpxA and ΔsrbA in by IR was readily reassembled in recGDR containing DrRecG and EcRecGQPW. hypoxia. Compared to WT, expression of hpxA was increased in ΔsrbA by These results suggest that the QPW residues facilitate strong binding of about 3-fold, while expression of srbA was reduced in ΔhpxA by about DrRecG to DNA junctions, thereby enhancing the efficiency of DrRecG in 0.6-fold. Our study suggests the presence of a putative linear genetic DNA repair process. relationship between two transcriptional regulators for hypoxia adaptation in A. nidulans. [Supported by the NRF (No. 2015R1D1A1A01057677)]

F010 F012 Yap1 and Skn7 are Involved in DNA Double-strand Break Complete Genome Sequencing of Clinical Isolated Repair by Homologous Recombination in Saccharomyces Salmonella enterica FORC_020 and Comparative Genome cerevisiae Analysis with S. enterica Typhimurium LT2 and S. enterica Newport USDA-ARS-USMARC-1972 Myung Ju Kim1,2, Dae Gwan Yi3, Jihyun Lee1,2, Ji Eun Choi1,2, Bohyun Park1, 1 1,2 Sujin In , and Woo-Hyun Chung * You-Tae Kim and Ju-Hoon Lee* 1College of Pharmacy, 2Innovative Drug Center, Duksung Women's Universiity Department of Food Science and Biotechnology, Institute of Life Sciences and 3Department of Biological Sciences, Seoul National University Resources, Kyung Hee University

Cells are continually exposed to numerous exogenous and endogenous Salmonella enterica is a well-known food-borne pathogen causing salmonellosis agents that cause DNA damage. Although substantial proportion of lethal according to what serotype could be harmful to human. S. enterica FORC_020 DNA damage is attributed by direct DNA strand breaks, oxidative DNA (NCCP P3567) is a clinical isolate from a blood sample of a food-poisoned adducts induced by reactive oxygen species (ROS) are also significant patient. To determine the serotype and understand its pathogenicity and sources of mutagenesis and genomic instability that might eventually lead virulence mechanism for food poisoning in genomic level, its genome was to tumorigenesis as well as aging. Pathways of DNA damage repair and completely sequenced using hybrid sequencing and analyzed using various oxidative stress responsive signaling have been proposed to be highly bioinformatic programs. The complete genome size of strain FORC_020 associated in the cell, but the underlying molecular mechanism remains was 4,799,793 bp with GC content of 52.26%. It contains 83 tRNA genes, 7 unknown. We employed mutant strains lacking Rad51, the homolog of E. rRNA operons, and a single additional 5S rRNA gene. The genome was coli RecA recombinase, and Yap1 or Skn7, two major transcription factors annotated to 4,671 open reading frames (ORFs) consisting of 3,957 ORFs responsive to ROS, to examine genetic interactions between double-strand predicted to encode functional proteins. Phylogenetical location of strain break (DSB) repair proteins and cellular redox regulators in budding yeast FORC_020 is closest to S. enterica Typhimurium LT2 in 16S rRNA sequence Saccharomyces cerevisiae. Abnormal expression of YAP1 or SKN7 aggravated analysis with >99% DNA sequence identity by BLASN program. In Average spontaneous mutation rate and sensitivity of rad51 mutant to DSB- and Nucleotide Identity (ANI) analysis and Multilocus Sequence Typing (MLST), ROS-generating reagents. Rad51 deficiency contributed to genome instability meanwhile, strain FORC_020 was located close to Newport Serotype and more in response to the increased ROS, and the accumulation of DSB belonged to Newport of MLST database. Comparative genomics was lesions raised the intracellular ROS level. Our findings suggest that there is performed using ACT and MAUVE and two large different regions were a significant crosstalk between DSB repair pathways and ROS signaling detected. One is for inner membrane protein clusters and the other is for proteins for cell survival and maintenance of genome stability in response proteins related to DNA sulfur modification. This region is unique in to genotoxic stresses. Salmonella as well as other bacteria. [Supported by grant from KRF] [Supported by grants from MFDS]

56 | www.msk.or.kr Poster

F013 F015 ABC Transporter Atm1 Plays Roles in Mitochondrial Functional Analysis of RraAS1 Interacting with the Catalytic Functions and Iron Homeostasis in Human Fungal Pathogen Domain of RNase ES Cryptococcus neoformans Daeyoung Kim, Sojin Seo, Boeun Lee, Minju Joo, Eunsoo Do, Se-Ho Park, and Won Hee Jung* Ji-Hyun Yeom, and Kangseok Lee* Department of Systems Biotechnology, Chung-Ang University Department of Life Science, Chung-Ang University

Iron sulfur cluster (ISC) is an indispensable cofactor for various enzymes, RNase E is an essential enzyme that participates in processing and including mitochondrial respiratory chains, DNA repair, transcription and degradation of RNAs in Escherichia coli. Enzymatic activity of RNase E is many catalytic enzymes. In addition, in the model yeast Saccharomyces controlled by a regulator of ribonuclease activity, RraA and RraB. The gram cerevisiae, the mitochondrial ABC transporter, Atm1, has an important positive bacterium Streptomyces coelicolor also contains homologs of role in exporting the unknown substrate for cytosolic ISC assembly machinery RNase E and RraA, designated as RNase ES, RraAS1 and RraAS2. In this for the cytosol and nucleus ISC containing proteins. In this study, we study, we investigated the inhibitory function of RraAS1 on ribonucleolytic identified an ortholog of S. cerevisiae Atm1 in the human fungal pathogen activity of RNase ES. Unlike other RraA homologs, which normally require Cryptococcus neoformans. To characterize the functions of Atm1 in C. both of the catalytic and scaffold domains, RraAS1 inhibited the enzymatic neoformans, the strain lacking ATM1 gene was constructed, and its activity of RNase ES in vivo and in vitro by interacting with the catalytic phenotypes related to mitochondrial functions and iron homeostasis domain of RNase ES, Coexpression of RraAS1 reduced the ribonucleolytic were investigated. Our results showed that activity of mitochondrial activity in cells overproducing RNase ES and consequently rescued these superoxide dismutase Sod2 was diminished and that cellular ROS was cells from growth arrest. Analyses of steady-state level of several RNase E highly increased in the atm1 mutant, which may be caused by distorted substrates indicated that the coexpression of RraAS1 efficiently inhibited mitochondrial ISC homeostasis in the mutant cells. Furthermore, we found RNase ES action and resulted in the increased abundance of each RNA that the atm1 mutant displayed increased intracellular iron level compared species. Intriguingly, deletion of rraAS1 in Streptomyces coelicolor resulted with wild-type. Overall, our results suggested that Atm1 influences mitochondrial in early sporulation and hyper-production of actinorhodin antibiotic functions and plays a role in iron homeostasis in C. neoformans. compared with wild type strain. These results suggest that RraAS1 affects [This study was supported by the Basic Science Research Program through the global RNA stability by regulating enzymatic activity of RNase ES in the National Research Foundation of Korea (NRF), funded by the Ministry Streptomyces coelicolor. of Science, ICT, and Future Planning NRF-2013R1A1A1A05007037]

F014 F016 Distinct Survival Strategies of Pseudomonas aeruginosa by Functional Analysis of RraAS2, a Streptomyces coelicolor Dual Promoters of the Major Catalase (KatA) Homolog of RraA

In-Young Chung, Bi-o Kim, Hye-Jung Jang, and You-Hee Cho* Jihune Heo, Sojin Seo, Boeun Lee, Daeyoung Kim, Minju Joo, Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Ji-Hyun Yeom, and Kangseok Lee* Sciences, CHA University Department of Life Science, Chung-Ang University

KatA is the major catalase required for hydrogen peroxide resistance and RraA is a protein inhibitor of RNase E, which catalyzes the endoribonucleolytic acute virulence in Pseudomonas aeruginosa PA14, whose transcription is cleavage of a large proportion of RNAs in Escherichia coli. RraA has also driven from the promoter (katAp1) located at 155 nucleotide (nt) upstream of been shown to regulate the activity of RNase ES, a functional Streptomyces the start codon. Here, we identified another promoter (katAp2), the +1 of coelicolor ortholog of RNase E, which has 58.0% identity to the amino-terminal which was mapped at the 51 nt upstream of the start codon, which was catalytic region of RNase E. The antibiotic-producing bacterium Streptomyces responsible for the basal transcription during the planktonic culture and coelicolor also contains homologs of RraA, designated as RraAS1 and down-regulated upon H2O2 treatment under the control by the master RraAS2. Here, we report that RraAS2 inhibits the endoribonucleolytic activity regulator of anaerobiosis, Anr. To dissect the roles of the dual promoters of RNase ES in vivo as well as in vitro; however, unlike other RraA homologs in conditions involving KatA, we created the promoter mutants for each that require the C-terminal scaffold domain to exert their inhibitory effect, -10 box (p1m, p2m, and p1p2m) and found the separate roles of the dual RraAS2 required both of two concurrent domains of the scaffold region promoters to cope with reactive oxygen and nitrogen species (ROS and and the catalytic domain of RNase ES to do so. We further show that RNS), which is dependent on the growth states. The katAp1 is required for deletion of the RraAS2 gene in S. coelicolor results in hyper-production of the function of KatA in the logarithmic growth phase as well as in acute actinorhodin antibiotic and a higher growth rate compared with wild type virulence, whereas katAp2 is required for the function of KatA in the strain. These findings suggest that RraAS2-mediated regulation of RNase stationary phase during the planktonic culture as well as in the prolonged ES activity contributes to modulating the cellular physiology of S. coelicolor. 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.

www.msk.or.kr | 57 2016 International Meeting of the Microbiological Society of Korea

F017 F019 RNase G Modulates Pathogenicity of Salmonella Typhimurium Molecular Mechanism of Anti-repressor by Ler to LEE5 through the Control of hns mRNA Abundance Promoter in Enteropathogenic Escherichia coli (EPEC)

Hong-Man Kim1, Daeyoung Kim1, Minho Lee1, Ji-Hyun Yeom1, Boeun Lee1, Minsang Shin1 and Hyon E. Choy2* 2 1 Wooseok Song , and Kangseok Lee * 1Department of Microbiology, Kyungpook National University School of Medicine 2 1Department of Life Science, Chung-Ang University Department of Microbiology, Chonnam National University Medical School 2Department of Microbiology, Catholic University of Daegu, School of Medicine Secretion of effector proteins in Enteropathogenic Escherichia coli (EPEC) Bacterial ribonucleases regulate gene expression through RNA processing and Enterohemorrhagic Escherichia coli (EHEC) is mediated by a specialized and decay. Among them, RNase G is an endoribonucleases, which is involved type III secretion system which components are encoded in the LEE1-5 in rRNA processing and degradation of a few mRNAs in Escherichia coli. operons. H-NS, a global repressor in E. coli, silences the expression of LEE However its physiological role remains largely uncharacterized. Here, we operons. Ler, a master regulator in LEE operons, shares 24% amino acid report that RNase G (Rng) controls expression levels of histone-like nucleoid identity and 44% amino acid similarity to H-NS. Interestingly, rather than a structuring protein (H-NS) encoded by hns, which were strongly associated gene silencer, its main role has been characterized as an antagonizing with the pathogenicity of S. Typhimurium cells in both epithelial cells and protein that relieves H-NS-mediated transcriptional silencing. In the previous mice. We observed that RNase G expression in S. Typhimurium cells was study we reported molecular mechanism for the repression of LEE5p in EPEC induced when they were exposed to high-salt condition and infected into and EHEC by H-NS as a protein–protein interaction between upstream epithelial cells, and coincidentally expression levels of hns was decreased. DNA-bound H-NS and the αCTD of promoter-bound RNAP. The mechanism We further demonstrated that expression levels of Salmonella virulence of Ler in alleviating genes repressed by H-NS is largely unknown. We regulators, hilA, sipA, and hns, were tightly regulated by RNase G in anoxic examined binding affinity by both regulator at LEE5 promoter using a high-salt and infection conditions, which has a correlation with S. Typhimurium various in vitro work. Our result show that binding affinity of Ler to the pathogenicity. In addition, we validated that hns mRNA abundance is LEE5p DNA is about 40 folds greater than that of H-NS using surface mediated by RNase G, where 5’UTR of hns mRNA was directly cleaved by plasmon resonance technology. Ler binding should remove H-NS binding RNase G in vivo and in vitro. In conclusion, we suggest that RNase G-mediated to the same stretch of DNA. modulation of Salmonella pathogenicity island 1 type III secretion system [Supported by grants from NRF-2014R1A2A1A10051664] involves H-NS as a key factor for the survival and virulence of S. Typhimurium in host cell.

F018 F020 Change of Biochemical Characteristics in aroA ompA Cloning and Expression of 5-aminolevulinic Acid Synthase Deletion in Salmonella enterica serovar Enteritidis Gene Involved in Secondary Metabolism of Kitasatospora cheerisanensis Kiju Kim and Tae-Wook Hahn* College of Veterinary Medicine & Institute of Veterinary Science, Kangwon Hwang Jae Yoon and Doo Hyun Nam* National University College of Pharmacy, Yeungnam University

Salmonella enterica serovars Enteritidis (SE) is one of the well-known Recently the biosynthetic pathway for the formation of 2-amino-3- foodborne pathogen causing salmonellosis in the world. Many attenuated hydroxycyclopent-2-enone (C5N) unit and its attachment to core structure Salmonella mutants were developed for using vaccine strain against of secondary metabolites in actinomycetes has been revealed. It has been salmonellosis and live attenuated Salmonella strains can be powerful revealed that three enzymes, 5-aminolevulinate synthase (ALAS), amino- vaccine candidates for expression of heterologous antigen to the mammalian levulinate-CoA ligase (ALL) and amide synthetase (AMS) are involved in immune system. In particular, antigen delivery using a Salmonella strains the formation and attachment of C5N unit in secondary metabolite, which can enhance mucosal immunity as well as systemic immune responses. appear as a set of encoded genes located within a relevant biosynthetic λ We constructed an aroA ompA deletion SE mutant using the phage -red gene cluster. Among the three genes, the ALAS gene of Kitasatospora recombinase system. The biochemical characteristics of SE mutant were cheerisanensis KCTC2395 which is assumed to be responsible for the C5N identified by the Vitek II system (bioMérieux, France) based on Gram-negative formation for the biosynthesis of bafilomycin B1 compound was cloned card according to manufacturer's instructions. The SE aroA ompA mutant and heterologously expressed in E. coli. The biochemical characterization resulted in an inability to produce H2S production (H2S), lipase (LIP) and of recombinant ALAS of K. cheerisanensis was attempted by colorimetric alpha-galactosidase (AGAL) compared with SE wild type. Interestingly, we assay of the enzyme activities in this study. The recombinant ALAS protein found the expression of another enzymes such as gamma-glutamyl- was confirmed to produce 5-ALA from glycine and succinyl-CoA. The enzyme transferase (GGT), L-lactate alkalinisation (ILATk) and phosphatase (PHOS) reaction occurred time-dependently upon the concentration of substrates. in SE aroA ompA mutant. These findings indicate that SE aroA ompA The optimal reaction pH was 8.5, and the optimal concentration of mutant may regulate to express different enzymes by unknown biochemical pyridoxal-5’-phosphate (PLP) as a cofactor in this reaction system was mechanism. found to be 0.1 mM.

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

58 | www.msk.or.kr Poster

F021 F022 The Velvet Regulators and Their Targets in Aspergillus Effects of the Number of the Origin of Replication on the Cell nidulans Physiology of Escherichia coli

Hee-Soo Park 1,3*, Pil Jae Maeng2, and Jae-Hyuk Yu3 Hee Jin Yang, Yuna Jung, and Jihyun F. Kim* 1School of Food Science and Biotechnology, Kyungpook National University, Department of Systems Biology, College of Life Science and Biotechnology, 2Department of Microbiology and Molecular Biology, Chungnam National Yonsei University University, 3Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA Bacteria, having circular genomes in most cases, have a single origin of replication (ori) for each replicon, while archaea and eukaryotes may have The NF-kB like fungal transcript factors VosA and VelB are key regulators multiple origins of replication in each chromosome. Initiation of DNA which control spore viability, maturation and β-glucan synthesis in replication in Escherichia coli is bi-directionally triggered at oriC and Aspergillus nidulans. The VosA-VelB hetero-complex directly or indirectly arrested at the termination sites (ter). We questioned what if E. coli has regulates spore-specific genes and development-associated genes. Here, multiple origins of replication in its chromosome and how it may affect cell we report various targets of the VosA-VelB hetero-complex. Frist, physiology including cell division. We attempted to introduce additional VosA-VelB controls a β-glucan synthesis in both asexual and sexual wild-type oriC of E. coli into intergenic regions that should have no direct spores. Second, VosA-VelB directly regulates trehalose biosynthesis in effect on cell physiology. Consequently, we constructed E. coli derivatives asexual spores. Third, mRNA accumulation of several developmental with two or three oriCs which are separated from the original oriC by 1 Mb genes is controlled by VosA and VelB. Overall, these results support that or 2 Mb. Recombinant E. coli cells exhibited a range of cell-size variations; the VosA-VelB complex is a master transcriptional unit for sporogenesis in some appeared indistinguishable from wild type with no morphological Aspergillus nidulans. 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.

www.msk.or.kr | 59 2016 International Meeting of the Microbiological Society of Korea

G001 G003 Metabolically Engineered Escherichia coli for Renewable Microbial Production of Gamma-butyrolactone via Production of 3-Aminopropionic Acid Metabolically Engineered Mannheimia succiniciproducens

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

In this study, a novel metabolic pathway was designed for the production γ-Butyrolactone (GBL) is an important four carbon (C4) chemical, which of 3-aminopropionic acid (3-AP) in Escherichia coli. Using a fumaric-acid has a wide range of industrial applications. GBL can be produced by acid producing E. coli strain as a host, the C. glutamicum panD gene (encoding treatment of 4-hydroxybutyric acid (4-HB), which is a derivative of succinic L-aspartate-α-decarboxylase) was overexpressed and the native promoter acid. Heterologous metabolic pathways were designed and established in of the aspA gene was replaced with the strong trc promoter, which allowed succinic acid overproducing M. succiniciproducens LPK7 by the introduction aspartic acid production through the aspartase (AspA)-catalyzed reaction. of heterologous genes that encode succinyl-CoA synthetase, CoA-dependent Additional overexpression of the aspA and phosphoenolpyruvate carboxylase succinate semialdehyde dehydrogenase, and either 4-hydroxybutyrate (ppc) genes, and the supplementation of ammonium sulfate in the medium dehydrogenase in LPK7 (p3S4CD) or succinate semialdehyde reductase in allowed production of 3.49 g/L 3-AP. This was further increased to 3.94 g/L LPK7 (p3SYCD). Fed-batch cultures of LPK7 (p3S4CD) and LPK7 (p3SYCD) by optimizing the expression level of PPC, which was achieved by evaluating resulted in the production of 6.37 and 6.34 g/L of 4-HB, respectively. 12 different combinations of synthetic promoters and RBS sequences. Finally, GBL was produced by acid treatment of the 4-HB obtained from Fed-batch culture of the final strain yielded 17.9 g/L 3-AP in 89 h, with an the fermentation broth. This study demonstrates that 4-HB, and potentially overall yield and productivity of 0.186 g 3-AP/g glucose and 0.200 g/L/h, other four carbon platform chemicals, can be produced by the engineered respectively. rumen bacterium M. succiniciproducens. [Development of systems metabolic engineering platform technologies [This work was supported by the Technology Development Program to for biorefineries(NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) Solve Climate Changes on Systems Metabolic Engineering for Biorefineries funded by the Ministry of Education, Science and Technology] from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557).]

G002 G004 Engineering TCA Cycle for Renewable Production of Fumaric Production of Tyrosine and Cadaverine by Metabolically Acid by Escherichia coli Engineered Escherichia coli Using Synthetic Small Regulatory RNA Tong Un Chae1, Chan Woo Song1,2, Dong In Kim1,3, Sol Choi1,2, 1 2 1 1,3,4 Jae Won Jang1,2, and Sang Yup Lee1,2,3* Ji Yeon Ha , Dokyun Na , Seung Min Yoo , and Sang Yup Lee * 1 1Metabolic and Biomolecular Engineering National Research Laboratory, Department Department of Chemical and Biomolecular Engineering (BK21 Plus Program), 2 KAIST, 2BioProcess Engineering Research Center, KAIST, 3BioInformatics Research of Chemical and Biomolecular Engineering (BK21 Plus program), KAIST, School of Integrative Engineering, Chung-Ang University, 3Bioinformatics Research Center, KAIST Center, KAIST, 4BioProcess Engineering Research Center, KAIST

Escherichia coli was metabolically engineered to produce a fumaric acid, Small regulatory RNAs (sRNAs) are gene expression regulators which act an intermediate of the TCA cycle. To redirect carbon flux through the on post-transcriptional phase of bacterial gene expression. Here, we glyoxylate shunt, the iclR gene was deleted, and the fumA, fumB, and developed synthetic sRNA system to down-regulate the gene expression. fumC genes were also deleted to enhance fumaric acid formation. The The target mRNA binding region of MicC, one of the well-studied sRNAs in resulting strain was able to produce 1.45 g/L of fumaric acid in flask Escherichia coli, was replaced to translation initiation sequence of our culture. Additionally, in silico flux response analysis was performed and target genes. We found that MicC scaffold based synthetic sRNA is the native ppc gene was overexpressed on plasmid level from tac promoter. properly repressed expression of DsRed2. Synthetic sRNAs was used for This resulting strain produced 4.09 g/L of fumaric acid. Moreover, the metabolic engineering to enhance the production of tyrosine and cadaverine. arcA and ptsG genes were deleted to reinforce the oxidative TCA cycle, Through screening of 14 different strains which harboring synthetic sRNAs, and the aspA gene was deleted to prevent the conversion of fumaric acid we isolated a tyrosine producer producing 2 g/L of tyrosine. Using a library into L-aspartic acid. For enhanced glucose uptake rate and fumaric acid of 130 synthetic sRNAs, we screened knockdown targets that increase productivity, the native promoter of the galP gene was replaced with trc cadaverine productivity substantially. promoter and the lacI gene was also deleted to avoid the use of inducer. [This work was supported by the Technology Development Program to Fed-batch culture of the final strain CWF812 produced 28.2 g/L fumaric Solve Climate Changes on Systems Metabolic Engineering for Biorefineries acid in 63 h with the overall yield and productivity of 0.389 g fumaric from the Ministry of Science, ICT and Future Planning (MSIP) through the acid/g glucose and 0.448 g/L/h, respectively. National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 [This work was supported by the Technology Development Program to and NRF-2012M1A2A2026557); the Intelligent Synthetic Biology Center Solve Climate Changes on Systems Metabolic Engineering for Biorefineries through the Global Frontier Project (2011-0031963) of the Ministry of from the Ministry of Science, ICT and Future Planning (MSIP) through the Science, ICT & Future Planning through the National Research Foundation National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 of Korea; the Commercializations Promotion Agency for R&D Outcomes and NRF-2012M1A2A2026557).] (COMPA-2015K000365) funded by the Ministry of Science, ICT and Future Planning (MISP).]

60 | www.msk.or.kr Poster

G005 G007 Production of Phenol from Glucose by Metabolically Native-sized Spider Silk Protein Production by Enhanced Engineered Escherichia coli Glycine Pool

Ji Yeon Ha1, Byoungjin Kim1, Hyegwon Park1, Dokyun Na2, and Sang Yup Lee1,3,4* Ji Yeon Ha1, Xiao-Xia Xia1, Do Kyun Na2, and Sang Yup Lee1,3,4* 1Metabolic and Biomolecular Engineering National Research Laboratory, Department 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), KAIST, 2School of Chemical and Biomolecular Engineering (BK21 Plus program), 2School of of Integrative Engineering, Chung-Ang University, 3Bioinformatics Research Integrative Engineering, Chung-Ang University, 3Bioinformatics Research Center, KAIST, 4BioProcess Engineering Research Center, KAIST Center, KAIST, 4BioProcess Engineering Research Center, KAIST

Engineering E. coli for biological production of phenol is considered infeasible Naturally found spider silk and elastin protein show unique characteristics due to its toxicity and complex biosynthetic network. To address these of highly repeated structure and extreme size as well as strength make challenges, we took advantage of the synthetic sRNA-based gene knockdown them attractive to many industrial applications. However, exceptional technology to simultaneously engineer 18 E. coli strains and screen them structure and size limits expression in heterologous hosts, where the for higher phenol tolerance and precursor tyrosine availability. The knock-down repetitive sequences in mRNA create extensive secondary structures. And of csrA and tyrR genes were identified to be crucial for redirecting carbon these structures decrease ribosome processivity and assist mRNA degradation. flux toward tyrosine and applied to all the E. coli strains with over-expression Using the naturally found protein, spider dragline silk protein, we present of the key genes for the biosynthesis of tyrosine and phenol (tyrosine techniques to solve biological problems that occurred: increasing the phenol-lyase). The engineered strains showed substantially different metabolic available ribosome pool and stabilizing mRNA for further degradation. Newly phenotypes. The engineered BL21(DE3) strain produced 419 mg/L of synthesized dragline silk protein produced increased titer than those reported phenol in flask culture and 1.69 g/L in fed-batch fermentation. A biphasic previously, therefore proving that the strategies used were efficient. The fed-batch fermentation using glycerol tributyrate was developed for in results provide insight into approaches to control translation efficiency of situ extraction while minimizing the phenol toxicity. The preferential proteins containing high molecular weight and highly repetitive sequence. partition of phenol from culture medium into glycerol tributyrate increased [This work was supported by the Technology Development Program to the titer and productivity to 3.79 g/L and 0.18 g/L/h, respectively. Solve Climate Changes on Systems Metabolic Engineering for Biorefineries [This work was supported by the Intelligent Synthetic Biology Center from the Ministry of Science, ICT and Future Planning (MSIP) through the through the Global Frontier Project (2011- 0031963) of the Ministry of National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 Science, ICT & Future Planning through the National Research Foundation and NRF-2012M1A2A2026557).] of Korea.]

G006 G008 Rapid Multiple Gene Knockout System Using Deletion of the Butyrate Kinase (buk) Gene is Essential for Integration-helper Plasmid the High Butyric Acid Selectivity in Clostridium acetobutylicum

Ji Yeon Ha1, Chan Woo Song1, and Sang Yup Lee1,2,3* Seon Young Park1, Yu-Sin Jang1, and Sang Yup Lee1,2* 1Metabolic and Biomolecular Engineering National Research Laboratory, Department 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), KAIST, of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for 2 2Bioinformatics Research Center, KAIST, 3BioProcess Engineering Research Systems and Synthetic Biotechnology, Institute, BioProcess Engineering Research Center, KAIST Center, Bioinformatics Research Center, Korea Advanced Institute of Science and Technololgy (KAIST) In this study, more rapid and efficient engineering method using integration helper plasmid in E. coli was developed. The integration helper plasmid, An important industrial chemical, butyric acid, has been widely used in pCW611, has two recombinases which are expressed in reverse direction food, pharmacy, animal feed supplement, and chemical industries. Butyric by two independent inducible systems. By using this system, required time acid producing Clostridium strains have a typical characteristic which is and effort can be significantly reduced because the iterative transformation of co-production of acetic acid with butyric acid. Thus, increasing the butyric the helper plasmid and curing steps are not required. We could delete one acid selectivity is important for economical butyric acid production. However, target gene in 3 days by using pCW611. To verify the usefulness of this increasing the butyric acid selectivity is difficult because of the complex gene manipulation system, the deletion experiments were performed for metabolic pathways and physiologies of clostridia. In this work, Clostridium knocking out four target genes individually (adhE, sfcA, frdABCD, and ackA) acetobutylicum was metabolically engineered to get the high butyric acid and two genes simultaneously for two cases (adhE-aspA and sfcA-aspA). Also, selectivity. The engineered C. acetobutylicum strain expressed the second fumaric acid producing E. coil strain was developed by deleting four target butyrate kinase of C. acetobutylicum encoded by bukII gene instead of genes (fumB, iclR, fumA, and fumC) in 10 days as a proof-of-concept study. butyrate kinase I encoded by buk gene. Moreover, metabolic pathways [This work was supported by the Technology Development Program to were further engineered to enhance the NADH-driving force. In batch Solve Climate Changes on Systems Metabolic Engineering for Biorefineries fermentation, the metabolically engineered C. acetobutylicum strain produced from the Ministry of Science, ICT and Future Planning (MSIP) through the 32.5 g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AA ratio) of National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 31.3 g/g from 83.3 g/L of glucose at pH 6.0. and NRF-2012M1A2A2026557)] [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)]

www.msk.or.kr | 61 2016 International Meeting of the Microbiological Society of Korea

G009 G011 Construction of Isopropanol-Butanol-Ethanol Production Production of 1-Propanol by Metabolically Engineered Platform in the Recombinant Clostridium Strains by L-threonine Overproducing Escherichia coli Metabolic Engineering Seon Young Park1, Yong Jun Choi1, Jin Hwan Park1, Seon Young Park1, Joungmin Lee1, Yu-Sin Jang1, and Sang Yup Lee1,2* Tae Yong Kim1, and Sang Yup Lee1,2,3* 1Metabolic and Biomolecular Engineering National Research Laboratory, Department 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for of Chemical and Biomolecular Engineering (BK21 program), BioProcess Engineering Systems and Synthetic Biotechnology, Institute, 2BioProcess Engineering Research Research Center, 2Center for Systems and Synthetic Biotechnology, Institute for Center, Bioinformatics Research Center, Korea Advanced Institute of Science and the Biocentury, 3Department of Bio and Brain Engineering and Bioinformatics Technololgy (KAIST) Research Center

1-Butanol is an attractive biofuel which can replace gasoline with its 1-Propanol is a greatly potential candidate of new biofuel which can specific properties. However, abnormal combustion can occur by its low substitute the gasoline. In this study, we introduced a novel pathway for octane rate. In this study, high amount of isopropanol-butanol-ethanol 1-propanol biosynthesis by overproducing L-threonine in the Escherichia (IBE) mixture was produced in the recombinant Clostridium acetobutylicum coli strain we have previously engineered. Formerly, Atsumi et al. produced strain by metabolic engineering. The hydG gene encoding putative B-593 3.5 g/L of 1-propanol by expressing Methanococcus jannaschii citramalate ferredoxin:NADP+ reductase was co-expressed with the adhB-593 gene encoding NADPH-dependent primary/secondary alcohol dehydrogenase, synthase (CimA) which directly converts pyruvate into 2-ketobutyrate in and IBE titer was further improved. The final strain, a host strain for a metabolically engineered E. coli. The E. coli TH20 strain which overproduce large-scale fermentation, was used to prove its possibility for industrial L-threonine, developed in our previous study, was further engineered in IBE production. Although many clostridial strains contain the orthologous this study. The flux response analysis for the carbon source usage optimization genes to hydGB-593 in their genome, C. acetobutylicum does not contain was conducted by in silico level, and then the knocking out of competing this gene. Our results showed that unlike other clostridial species, C. pathway was followed. The result strain succeeded in producing 1-propanol acetobutylicum might have a different mechanism of NADPH regeneration and the additional strain engineering further increased the titer. and the expression of hydGB-593 might increase the NADPH regeneration in [This work was supported by the Advanced Biomass R&D Center (ABC) of C. acetobutylicum. Global Frontier Project funded by the Ministry of Science, ICT and Future [This work was supported by the Technology Development Program to Planning (ABC-2011-0031350). Systems Metabolic Engineering work to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries Solve Climate Change was supported by Biorefineries from the Ministry of from the Ministry of Science, ICT and Future Planning (MSIP) through the Science, ICT and Future Planning (MSIP) through the National Research National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1- and NRF-2012M1A2A2026557) and by the C1 Gas Refinery Project funded A2A2026557).] by the MSIP through the NRF of Korea (2015M3D3A1A01064918)] G010 G012 Metabolic Engineering of Escherichia coli for Short Chain Production of Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) Alkanes Production by Metabolically Engineered Escherichia coli

Seon Young Park1, Yong Jun Choi1, and Sang Yup Lee1,2,3* Kyeong Rok Choi1, Jung Eun Yang1, Yong Jun Choi2, Seung Hwan Lee3, 4 5 1 1Metabolic and Biomolecular Engineering National Research Laboratory, Department Bong Keun Song , Si Jae Park , and Sang Yup Lee * of Chemical and Biomolecular Engineering (BK21 program), BioProcess Engineering 1Metabolic and Biomolecular Engineering National Research Laboratory, Department Research Center, 2Center for Systems and Synthetic Biotechnology, Institute for of Chemical and Biomolecular Engineering (BK21 plus Program), BioProcess the Biocentury, 3Department of Bio and Brain Engineering and Bioinformatics Engineering Research Center, KAIST, 2School of Environmental Engineering, Research Center University of Seoul, 3Dept. of Biotechnology and Bioengineering, Chonnam National University, 4Chemical Biotechnology Research Center, Korea Research So far, there have been many efforts on biofuel productions in microbes. Institute of Chemical Technology, 5Department of Environmental Engineering However the production of highly demanded fuels like gasoline has not and Energy, Myongji University been achieved. In this research, Escherichia coli was developed for producing short-chain alkanes (gasoline), free fatty acids (FFAs), fatty esters and fatty Polyhydroxyalkanoates (PHAs), polyesters accumulated in many bacteria, alcohols. To prevent β-oxidation which degrades fatty acyl-CoAs generated has received great interest because of their unique characteristics, biode- in vivo, the fadE gene was deleted. For increasing the initiation of fatty gradable and biocompatible thermoplastic. Among various PHA copolymers, acid biosynthesis, the fadR gene was knocked out and that consequently poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] is an important promoted the activity of FebH, 3-oxoacyl-ACP synthease and reduced the copolymer because of its lower melting point and much better flexibility. formation of unsaturated fatty acyl-ACPs. Additionally, for conversion of So far, secondary carbon source was required to produce P(3HB-co-3HV). short chain fatty acyl-ACPs to the corresponding FFAs, a mutant TesA was The toxicity of the auxiliary carbon source, however, retards cell growth conducted. The serial reactions of fatty acyl-CoA synthetase from E. coli, while enhancing P(3HB-co-3HV) production. Here, we developed an E. coli fatty acyl-CoA reductase from Clostridium acetobutylicum and fatty aldehyde stably synthesizing 3HB-CoA and 3HV-CoA in controlled ratio from glucose decarbonylase from Arabidopsis thaliana converted the short chain FFAs without feeding of exogenous auxiliary carbon source by metabolic engineering. into corresponding alkanes. Consequently, the final strain produced 580.8 Two different metabolic pathways for the production of propionyl-CoA mg/L of short chain alkanes. from 2-ketobutyrate were constructed by introducing a series of heterologous [This work was supported by the Advanced Biomass R&D Center(ABC) of genes into E. coli. The engineered strains were proven to efficiently synthesize Global Frontier Project funded by the Ministry of Science, ICT and Future P(3HB-co-3HV) independent of exogenous auxiliary carbon source. Planning (ABC-2011-0031350). Systems Metabolic Engineering work to [This work was supported by the Technology Development Program to Solve Climate Change was supported by Biorefineries from the Ministry of Solve Climate Changes on Systems Metabolic Engineering for Biorefineries Science, ICT and Future Planning (MSIP) through the National Research from the Ministry of Science, ICT and Future Planning (MSIP) through the Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1- National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 A2A2026557).] and NRF-2012M1A2A2026557).]

62 | www.msk.or.kr Poster

G013 G015 Amino Acid L-arginine Production in a Metabolically L-Ornithine Bioproduction in Metabolically Engineered Engineered Corynebacterium glutamicum Corynebacterium glutamicum

Kyeong Rok Choi1, Seok Hyun Park1, Hyun Uk Kim1,2, Tae Yong Kim1,2, Kyeong Rok Choi1, Seo Yun Kim1, Joungmin Lee1, and Sang Yup Lee1,2,3* 3 3 1,2,4 Jun Seok Park , Suok-su Kim , and Sang Yup Lee * 1Metabolic and Biomolecular Engineering National Research Laboratory, Department 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus rogram), Center for 2 of Chemical and Biomolecular Engineering (BK21 Plus rogram), Center for Systems and Synthetic Biotechnology KAIST, BioInformatics Research Center, 3 Systems and Synthetic Biotechnology KAIST, 2BioInformatics Research Center, KAIST, BioProcess Engineering Research Center, KAIST KAIST, 3Daesang Corporation Research Center, 4BioProcess 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 Metabolic engineering approach was applied to Corynebacterium glutamicum engineered Corynebacterium glutamicum ATCC 13032. To block competitive for L-arginine production. To make C. glutamicum tolerant to L-arginine, branch pathway and the conversion of L-ornithine to citrulline, proB and random mutagenesis was first performed. Subsequently, arginine operon argF were first deleted, respectively. The L-arginine operon regulatory repressor proteins were inactivated, and the PPP flux was strengthened by repressor gene argR was also removed to enhance the flux toward ornithine, downregulating pgi and overexpressing opcA, pgl, tal, tkt, and zwf. Then, resulting in 230 mg/L of L-ornithine from glucose in flask culture. Further Ncgl1221 encoding L-glutamate exporter was inactivated to channel engineering by plasmid-based overexpression of argCJBD genes from an L-glutamate to L-arginine formation. Additionally, argF and carAB expression arginine overproducer C. glutamicum ATCC 21831 resulted in the 7.19 g/L levels were optimized for converting L-ornithine to L-citrulline effectively, L-ornithine production. In effort to enrich the NADPH pool, the carbon flux followed by argGH operon overexpression. Fed-batch fermentation of the was redirected towards the pentose phosphate pathway by changing the final strain was performed in a 1,500 L bioreactor resulting 81 g/L of start codons of the pgi and zwf genes and replacing the native promoter of L-arginine production. The approaches described here will be useful in the tkt operon with the strong sod promoter. Fed-batch cultivation of the developing corynebacteria regarding the production of arginine and its final strain YW06 (pSY223) achieved 51.5 g/L L-ornithine titer in 40 h with derivatives. the overall productivity of 1.29 g/L/h. The results demonstrate the efficient [This work was supported by the Technology Development Program to L-ornithine production in metabolically engineered C. glutamicum. Solve Climate Changes on Systems Metabolic Engineering for Biorefineries [This work was supported by the Technology Development Program to from the Ministry of Science, ICT and Future Planning (MSIP) through the Solve Climate Changes on Systems Metabolic Engineering for Biorefineries National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 from the Ministry of Science, ICT and Future Planning (MSIP) through the and NRF-2012M1A2A2026557).] National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557).]

G014 G016 Cadaverine Overproduction in a Metabolically Engineered Engineering of Metabolic Flux into Novel Escherichia coli Cell Factory Gamma-aminobutyric Acid Production Pathway by Introduction of Synthetic Scaffolds in Escherichia coli Kyeong Rok Choi1, Zhi-Gang Qian1, Hye Min Park1, and Sang Yup Lee1,2,3* 1Metabolic and Biomolecular Engineering National Research Laboratory, Department Van Dung Pham, Sivachandiran Somasundaram, and Soon Ho Hong* of Chemical and Biomolecular Engineering (BK21 Plus rogram), Center for Department of Chemical Engineering, University of Ulsan Systems and Synthetic Biotechnology KAIST, 2Institute for the BioCentury, KAIST, 3 BioProcess Engineering Research Center, KAIST In general gamma-aminobutyric acid (GABA) pathway involves the decar- boxylation of glutamate, which is produced from sugar by Corynebacterium A five-carbon diamine cadaverine can be used as a building block for fermentation. GABA can be used for the production of pharmaceuticals sustainable polymer production. Bio-based production of cadaverine from and functional foods. Due to the increasing demand of GABA, it is essential renewable carbon sources is a promising and sustainable alternative to to create an effective alternative pathway for the GABA production. In this the current petrochemical production. Here, we report a series of metabolic study, Escherichia coli were engineered to produce GABA from glucose via engineering on Escherichia coli for the production of cadaverine from GABA shunt, which consists of succinate dehydrogenase, succinate-semialdehyde glucose in defined medium. To enrich cadaverine, degradative pathways dehydrogenase and GABA aminotransferase. The three enzymes were on the target product were blocked. The engineered strain produced 9.61 physically attached each other through synthetic scaffold, and the Krebs g/L of cadaverine with a productivity of 0.32 g/L/h by fed-batch cultivation. cycle flux was redirected to GABA pathway. By introduction of synthetic [This work was supported by the Technology Development Program to scaffold, 0.75 g/L of GABA was produced from 10 g/L of glucose at 30oC Solve Climate Changes on Systems Metabolic Engineering for Biorefineries and pH 6.5. The inactivation of competing metabolic pathways provided from the Ministry of Science, ICT and Future Planning (MSIP) through the 15.4% increase of the final GABA concentration. National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 [This work was supported by a grant from the Next-Generation BioGreen and NRF-2012M1A2A2026557).] 21 Program (SSAC, grant number: PJ011116) by RDA, and Basic Science Research Program by the Ministry of Education (NRF-2014R1A1A20- 54726).]

www.msk.or.kr | 63 2016 International Meeting of the Microbiological Society of Korea

G017 G019 Construction of Acidic Amino Acids Sensing Escherichia coli Isolation of Biogenic Amine Non-producing Bacillus subtilis by Introduction of Chimeric Two-component System SCM121 and Medium Optimization for Improving Biomass by Response Surface Methodology Murali kannan Maruthamuthu and Soon Ho Hong* Department of Chemical Engineering, University of Ulsan Su-Ji Jeong, Hee-Jong Yang, Seong-Yeop Jeong, Jeong Seon Eom, and Do-Youn Jeong* In an attempt to create an acidic amino acid-sensing Escherichia coli, a Microbial Institute for Fermentation Industry (MIFI) chimeric sensor kinase (SK)-based biosensor was constructed using Pseudomonas putida AauS. AauS is a sensor kinase that ultimately controls expression of Biogenic amines are produced primarily by microorganisms found in fermented the aau gene through its cognate response regulator AauR, and is found foods and are often implicated seriously poisoning in the body of human. only in P. putida KT2440. The AauZ chimera SK was constructed by integration Biogenic amines are thus considered a risk for human health. 620 strains of the sensing domain of AauS with the catalytic domain of EnvZ to control were isolated from Korean traditional fermented food in Sunchang and the expression of the ompC gene in response to acidic amino acids. Real-time investigated biochemical characterization and the ability of biogenic amines quantitative PCR and GFP fluorescence studies showed increased ompC non-producing. SCM121 was selected based on the various properties gene expression and GFP fluorescence as the concentration of acidic amino such as extracellular enzyme, antioxidant and antimicrobial activities. The acids increased. These data suggest that AauS-based recombinant E. coli strain SCM121 was named as Bacillus subtilis SCM121 by 16S rRNA sequencing can be used as a bacterial biosensor of acidic amino acids. By employing and biochemical characterization. And then, we investigated cell growth the chimeric SK strategy, various bacteria biosensors for use in the for application of industrial field, and optimized the culture medium constituents development of biochemical-producing recombinant microorganisms can using response surface methodology. Plackett-Burman experimental design be constructed. was used for screening of medium constituent, and sucrose, K2HPO4, and [This work was supported by a grant from the Next-Generation BioGreen MgSO4 were selected as important factors for improving cell growth. In 21 Program (SSAC, grant number: PJ011116) by RDA, and Basic Science order to find out optimal concentration on selected constituents, we carried Research Program by the Ministry of Education (NRF-2014R1A1A20- out central composite design. Consequently, optimal concentrations of 54726).] 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).]

G018 G020 3D Wave of Fermented Soybean Extracts Suppress Isolation of Bacillus subtilis SCM146 Having Antimicrobial Proliferation of Human Breast Cancer MDA-MB-231 Cells Activity and Medium Optimization for Improving Biomass by Response Surface Methodology Jameon Park1,2 and Han Bok Kim1,2* 1Department of Biotechnology, Hoseo University, 2The Research Institute for Hee-Jong Yang, Su-Ji Jeong, Seong-Yeop Jeong, Basic Sciences, Hoseo University Jeong Seon Eom, and Do-Youn Jeong* Microbial Institute for Fermentation Industry (MIFI) 3D wave part of fermented soybean extracts can be separated from matter of fermented soybean extracts. Fermented soybean extracts inhibit proliferation 620 strains were isolated from Korean traditional fermented food in of human breast cancer MDA-MB-231 cells. In this study it is demonstrated Sunchang and investigated biochemical characterization and the ability of that 3D wave of fermented soybean extracts could be transferred to water, biogenic amines non-producing. SCM146 was selected based on the various and the water inhibited proliferation of human breast cancer MDA-MB-231 properties such as extracellular enzyme, antioxidant activities, and et al., cells. Drug delivery into brain cells will be possible using 3D wave of its and was named as B. subtilis SCM146 by 16S rRNA sequencing and matter. biochemical characterization. And then, we investigated cell growth for Keywords: 3D wave, fermented soybean, breast cancer 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).]

64 | www.msk.or.kr Poster

G021 G023 Isolation of Biogenic Amine Non-producing Lactobacillus KCl Effect on Growth of Leuconostoc mesenteroides CS-5 brevis SCML67 and Medium Optimization for Improving under the High Salt Concentration Biomass Using Statistical Technique Oh Sik Kwon Su-Ji Jeong, Hee-Jong Yang, Seong-Yeop Jeong, Major in Biological Science, College of Natural Science, Keimyung University Jeong Seon Eom, and Do-Youn Jeong* Microbial Institute for Fermentation Industry (MIFI) Characteristics of Leuconostoc mesenteroides strains isolated from chonggak kimchi was studied to identify salinity nature. Among the isolates, a strain 583 strains were isolated from Korean traditional fermented food in Sunchang named as CS-5 showed an optimal growth temperature that ranged from and investigated biochemical characterization and the ability of biogenic 24°C to 28°C. Interestingly the CS-5 was turned out to be a dextran amines non-producing. SCML67 was selected based on the various properties producing strain. It could grow in the media containing 6% NaCl. At 5% such as extracellular enzyme, antioxidant activities, and ability of biogenic salinity the CS-5 revealed much better growth in media containing glucose amine non-producing, and named as L. brevis SCML67 by 16S rRNA sequencing. and fructose as a monosaccharide than a strain of CL-1 that could not grow And then, we investigated cell growth for application of industrial field, at all. From tests of disaccharides (maltose, sucrose), the CS-5 showed strong and optimized the culture medium constituents using response surface growth nature at 6% salinity in which the CL-1 could not grow at all. This methodology. Plackett-Burman experimental design was used for screening kind of salt tolerance was also appeared in the media containing trehalose, of medium constituent, and molasses, CSP, and urea were selected as melibiose and cellobiose that were known to be hard to ferment. Sole important factors for improving cell growth. In order to find out optimal addition of KCl (40 mM, 54 mM) on growth media for CS-5 showed small concentration on selected constituents, we carried out central composite increase for its cell growth determined by pH of supernatant from fermented design. Consequently, optimal concentrations of molasses, CSP, and urea media. However, addition of 40 mM KCl in media containing 4% to 8% were predicted to be 1.5%, 2.0%, and 1.0%, respectively. By the model NaCl enhanced growth of CS-5 dramatically. This implies that higher salt verification, we confirmed about 1.3-fold improvement of the dried cell concentration can be overcome by addition of KCl during cell growth of weight from 2.457±0.006 g/L to 3.1088±0.0338 g/L when compared to CS-5 as lactic acid bacteria. From a test for dextran production, the CS-5 basal medium. Finally, we carried out experiments for establishing optimal showed higher values of viscosity (0.153 Pa.sn) in media containing 30% pH and culture temperature, confirmed maximal cell growth 4.12±0.0985 sucrose (w/v) comparing to the controls (0.01 Pa.sn). g/L at 37°C and initial pH 7.0. Keywords: Dextran, Chonggak Kimchi, Leuconostoc mesenteroides, Salinity [Supported by High value-added Food Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries (No. 313037-3).]

G022 G024 Isolation of Biogenic Amine Non-producing Lactobacillus Oxidation of Methane throuth Component Interactions brevis SCML458 Having Antioxidant Activity and Medium from Type II Methanotrophs Optimization for Improving Biomass Min Young Song1, Eun Taek Seol1, and Seung Jae Lee1,2* Hee-Jong Yang, Su-Ji Jeong, Seong-Yeop Jeong, 1Department of Chemistry, Chonbuk National University 2 Jeong Seon Eom, and Do-Youn Jeong* Research Institute of Physics and Chemistry, Chonbuk National University Microbial Institute for Fermentation Industry (MIFI) Methane hydroxylation through methane monooxygenases (MMOs) is a key aspect due to their control of the carbon cycle in the ecology system Antioxidant are substances that may protect cells from the damage and recent applications of methane gas in the field of bioenergy and caused by unstable molecules known as free radicals. Free radical damage bioremediation. Methanotropic bacteria perform a specific microbial conversion may lead to cancer. In this study, 583 strains were isolated from Korean from methane, one of the most stable carbon compounds, to methanol through traditional fermented food in Sunchang and investigated biochemical elaborate mechanisms. MMOs express particulate methane monooxygenase characterization. SCML458 was selected based on antioxidant activities (pMMO) in most strains and soluble methane monooxygenase (sMMO) and ability of biogenic amine non-producing, and named as L. brevis SCML458 under copper-limited conditions. The mechanisms of MMO have been widely by 16S rRNA sequencing. And then, we investigated cell growth for application studied from sMMO included in the bacterial multicomponent monooxygenase of industrial field, and optimized the culture medium constituents using (BMM) superfamily. Mechanism studies of sMMO have been intensively response surface methodology. Plackett-Burman experimental design was studied by the supports of advanced biophysics, especially in the Methylococcus used for screening of medium constituent, and protease peptone, CSP, capsulatus (Bath) and Methylosinus trichosporium OB3b strains. Structural and yeast extract were selected as important factors for improving cell studies of three components of sMMO, a hydroxylase (MMOH), a regulatory growth. In order to find out optimal concentration on selected constituents, component (MMOB), and a reductase (MMOR), have provided crucial we carried out central composite design. Optimal concentrations of protease information about their catalytic activities. In this study, we report successful peptone, CSP, and yeast extract were predicted to be 2.0%, 2.5%, and growing and expression from type II methanotrophs, Methylosinus sporium 2.0%, respectively. By the model verification, we confirmed about 1.3-fold and Methylocystis sp. Strain M. improvement of the dried cell weight from 2.40±0.01 g/L to 2.97±0.05 g/L [Supported by grant from KRF of Korea.] 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).]

www.msk.or.kr | 65 2016 International Meeting of the Microbiological Society of Korea

G025 G027 Stability and Inactivation Mechanism of Porcine Parvovirus Bioconversion Efficiency in Accordance with the Substrate against High Temperature Treatment Concentrations

Sang Eun Han1, Jung Eun Bae2, and In Seop Kim1* Sung-Ho Joo, Won Mun Kim, Kwang-Su Lee, and Ki-Sung Lee* 1Department of Biological Sciences and Biotechnology, Hannam University Department of Biology & Medicinal Sciences, Pai Chai University 2BioPS We carried out experiments related with bioconversion efficiency according Viral contamination of human plasma or mammalian cell cultures in GMP to the higher concentrations of substrate (Arg;Arginine). Of the biofunctional manufacturing facility represents a serious safety threat to biopharmaceutical resources, emphases are placed on physio-biochemically important amino industry. Such adverse events usually require facility shutdown for cleaning/ acids such as ornithine (Orn), citrulline (Cit) and r-aminobutyric acid decontamination, and thus result in significant loss of production and/or (GABA). We isolated six strains (KA111, KA115, KA182, KA198, KA217, and delay of product development. Therefore, treatment with steam and/or KA222), which were tested and analyzed bioconversion activities under dilute NaOH are commonly used to disinfect manufacturing vessels and changing the concentrations of Arg at the higher levels of 3 times (3X) and tools in the biopharmaceutical industry. Porcine parvovirus (PPV) has 5 times (5X). As the results, when we applied high concentration of Arg been known to show the highest resistance to a range of physico-chemical with 3 times (3X) or 5times (5X) that is higher than the normal condition treatment. PPV is the best model virus for cleaning validation. In this study, (control group in the media), KA182, KA198, KA217, and KA222 did we examined inactivation kinetics of PPV against high temperature treatments convert Arg into Orn and/or Cit more than amounting to 70% in complex (70, 80, 90°C) in order to optimize the thermal treatment process to medium. But in the minimal medium only KA222 strain did convert Arg inactivate viral contaminants. PPV showed resistance against 90 min into Orn and/or Cit. On the other hand, KA111, KA115 did not covert. treatment at 70°C and 80°C with the log reduction value of 1.44 and 3.91, Finally, multi-biofunctional microbes (KA111, KA115, KA182, KA198, KA217, respectively. PPV was completely inactivated after 20 min treatment at and KA222) were related to biodegradability and bioconversion activity. 90°C. Thermal inactivation mechanism was investigated by quantitative According to our results, we suggest that our identified bio-functional real-time PCR determination of leakage of DNA using endonuclease microbes had effect on bio-ethanol process, bioremediation and regeneration treatment to thermally inactivated PPV as well as intact PPV. From this of polluted waste materials. study, it was found that thermal inactivation at 90°C was not due to [This work was supported by the Human Resource Training Program for disintegration of the viral capsid. Further study to elucidate the thermal Regional Innovation and Creativity through the Ministry of Education and inactivation mechanism of PPV is currently under way. National Research Foundation of Korea (2015035949).]

G026 G028 Biofunctionality and Bioconversion Activities of KA111 Characterization and Validation of the Modified tuf (Pseudomonas fragi), KA115 (Bacillus methylotrophicu), Promoter to Develop the Efficienct Recombinant Protein KA182 (Bacillus subtilis), KA198 (Bacillus safensis), KA217 Expression Vector System for Lactococcus lactis IL1403 (Bacillus pumilus), KA222 (Bacillus licheniformis) Inseon Kim1,2, Jinduck Bok2, Sangkee Kang2, 1 1 Sung-Ho Joo, Won Mun Kim, Kwang-Su Lee, and Ki-Sung Lee* Chongsu Cho , and Yunjaie Choi * 1 Department of Biology & Medicinal Sciences, Pai Chai University Department of Agricultural biotechnology, Seoul National University 2Institute of Green-Bio Science & Technology, Seoul National 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 Lactococcus lactis (L. lactis) has been known to be a good recombinant resources and to elucidate new biofunctionalities and bioconversion activities host for expression of recombinant proteins with ease of manipulation of six kinds of microorganisms, we performed the following experiments. and a live oral vaccine carrier. Therefore, many approaches have been These biofunctional resources are involved in physio-biochemically important attempted to improve the recombinant protein in L. lactis. To improve the amino acids such as ornithine (Orn), citrulline (Cit) and r-aminobutyric acid level of protein expression in this study, we developed strong promoters (GABA). We isolated these strains (KA111, KA115, KA182, KA198, KA217, for L. lactis by modifying the tuf promoter (189 bp) which was previously and KA222), which were tested and analyzed bioconversion activities studied by our group. The tuf promoter was modified by repeating core transforming Arg (arginine) into Orn and Cit. As the results, all of the six region (119 bp) such as -35 and -10 cis-acting elements to recruit the strains showed bioconversion activities converting Arg into Orn and Cit transcriptional initiation factors and altering the ‘G’, ‘C’ sequences to the directly or indirectly. ‘A’ at downstream of ribosome binding site. We successfully constructed Also, their bioconversion activities showed very strong regardless of complex mtuf1, mtuf2, mtuf3, mtuf1-1, and mtuf2-1 promoters without superfluous medium, malt extract medium composed of natural product, but those sequences by serial PCR steps. For validation of the efficacy of modified showed weak in the minimal medium. All of the six strains could bioconvert promoters, we carried out luciferase assay, qRT-PCR, western blot, and in Arg into Orn and/or Cit almost 100% efficiently not only on the complex vivo imaging. We have found that mtuf1, mtuf2, and derivative mutf2-1 medium but also on MEM. On the other hand, in the case of on the promoters expressed higher level of protein than original tuf promoter. In minimal medium, five strains (KA115, KA182, KA198, KA217, and KA222) specific, mtuf2-1 has shown great potential for application in construction could bioconvert Arg into Orn more than 50%, but KA111 strain couldn’t of recombinant L. lactis as oral delivery vehicle. bioconvert. [Supported by Korea Institute of Planning and Evaluation for Technology [This work was supported by the Human Resource Training Program for in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Regional Innovation and Creativity through the Ministry of Education and Technology Development Program, funded by Ministry of Agriculture, National Research Foundation of Korea (2015035949).] Food and Rural Affairs (MAFRA)(115084-2)]

66 | www.msk.or.kr Poster

G029 G031 Inhibition of Enterotoxigenic Escherichia coli Cell-growth by CTHRC1 Protein Binding RNA-aptamer Selection by SELEX ETEC Specific Binding DNA Aptamer Hee-Young Cho1, Woo-Ri Shin1, Kyeong-Ah Lee1, Se Hee Lee1, 1 1 2 Woo-Ri Shin1, Sang-Hee Lee1, Ji-Young Ahn1, Jiho Min2, and Yang-Hoon Kim1* Simranjeet Singh Sekhon , Ji-Young Ahn , Dae-Ghon Kim , 3 1 1 Jiho Min , and Yang-Hoon Kim * Department of Microbiology, College of Natural Sciences, Chungbuk National 1 University, 2Graduate School of Semiconductor and Chemical Engineering, Chonbuk Department of Microbiology, College of Natural Sciences, Chungbuk National 2 National University University, Research Institute of Clinical Medicine of Chonbuk National University, 3Graduate School of Semiconductor and Chemical Engineering, Enterotoxigenic Escherichia coli (ETEC) strains may cause diarrheal diseases Chonbuk National University in humans and farming animals. For pigs and calves, K88 and K99 fimbriae, which are expressed by ETEC strains, are responsible for most neonatal Hepatocellular carcinoma (HCC) is the most frequent type of liver cancer. infections and the majority of diarrheal infections. Aptamer is single-stranded Alpha-fetoprotein (AFP) is a popular biomarker for HCC, however AFP is DNA or RNA that can bind to various targets with high specificity. It has a not detected in first stage. Collagen Triple Helix Repeat Containing 1 number of advantages like high stability at high temperature and pH easily (CTHRC1) protein is overexpressed in HCC, which acceleration tumor cell modified with various functional groups. In this study, we generated DNA invasion. The reason we use aptamer to detect CTHRC1 protein is aptamer’s aptamers specific to ETEC (K88, K99 strains) using a whole bacterial many advantages. Aptamer have less sensitive to high temperature, various Cell-SELEX process and characterized their affinity, and specificity to other application and high affinity to their target. Generated RNA aptamer cells. These aptamers modulate processed bacterial cell growth rate in candidates were using Systematic Evolution of Ligands by EXponential ETEC K88 & K99. enrichment (SELEX) process. 32 RNA aptamer candidates were selected by [This work was carried out with the support of "Cooperative Research real-time PCR and T-vector cloning. Followed by Surface Plasmon Resonance Program for Agriculture Science & Technology Development (Project title: (SPR) process to obtain high affinity aptamer which is specific binding to Development of Monitoring and Diagnostic Method for Environmental CTHRC1 protein. These aptamers can be treated in human Hepatocellular Animal Disease, Project No: PJ010530)" Rural Development Administration, carcinoma cell lines. The objective of our research to reduce the sizes of Republic of Korea.] 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).]

G030 G032 Human Immunodeficiency Virus Type 1 Safety of Blood Isolation and Identification of Strain for Saccharina japonica Coagulation-related Plasma Products Fermentation

Jung Eun Bae, Eun Kyo Jeong, Dong Joo Yu, Da Jeong Kim, Jung Sun Jeong, Hyeon Song Oh and Youn Tae Chi* Sang Eun Han, and In Seop Kim* School of Biological Sciences and Technology, Chonnam National University Department of Biological Sciences and Biotechnology and Center for Biopharmaceuticals Safety Validation, Hannam University Saccharina japonica is mainly distributed in the coast of East Asia, including Korea, China and Japan. Saccharina japonica contains large The production of plasma derivatives of human origin must take into amounts of functional material, such as alginate and fucoidan. Also it account the possible presence of pathogenic viruses in the original includes various inorganic salts such as calcium, magnesium and iodine. material. Thus, special precaution must be taken during the production to The main component of Saccharina japonica, alginate, has several functions assure against the possibility of the products transmitting infectious such as obesity inhibition, constipation relief, anti-cholesterol effect, disease to the recipients. The most dangerous blood-borne virus of clinical detoxification and heavy metal emission. In addition, α-D-mannuronic concern is human immunodeficiency viruses type I (HIV-1). However, acid and β-L-guluronic acid formed from depolymerization of alginate are there has been limited information about HIV safety for fibrin glue, effective in anti-cancer and anti-bacterial actions. However, breakdown antithrombin III, and factor IX. This study was thus designed to evaluate and absorption of alginate do not occur efficiently in the human body. the HIV-1 inactivation efficacy of manufacturing processes for fibrin glue, Fermentation can be one of the solution for this limitation. Therefore, in antithrombin III, and factor IX. Solvent/detergent (S/D) and 60℃ heat this study, we aimed to find the best strains for fermenting Saccharina treatments during the manufacture of fibrin (fibrin glue syringe I) have japonica. 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.

www.msk.or.kr | 67 2016 International Meeting of the Microbiological Society of Korea

G033 G035 Effect of Before and After Fermented Dendropanax Isolation and Identification of Ground Microorganism to morbifera Extract on HEK293 and PANC-1 Cells Find New Beneficial Microbial Agents

Ra Min Seo and Youn Tae Chi* Jin Sung Kim and Youn Tae Chi* School of Biological Sciences and Technology, Chonnam National University School of Biological Sciences and Technology, Chonnam National University

Dendropanax morbifera is a genus of flowering plants in the family We isolated and identified micro organism from ground sample of Chonnam Araliaceae. Today, it attracts people’s attention for positive effects on area to find new beneficial microbial agents. We characterized the identified human health so that many studies have been carried out and are still new micro organisms and examined their anti-microbial activity for plant ongoing. Several studies found extracts from Dendropanax morbifera have disease. In addition, antibiotics extracted from the micro organisms was enormous implications including anti-cancer and anti-bacterial activity. tested to find out the best condition for production. Through this study, However, there is limitation that all these beneficial compounds the plant we found that the identified ground micro organisms can be used for has are not assimilated into the human body. Fermentation is suggested potential beneficial microbial agents. 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 G036 Comparing the Ingredients and Antioxidant Effects of Screening and Application of Xylanase Producing Dendropanax morbifera Fermentation Using Microorganism Sphigobacterium sp. A10 Strain

Ho Min Song, Sang Hoon Na, and Youn Tae Chi* Sang Hoon Na and Youn Tae Chi* School of Biological Sciences and Technology, Chonnam National University School of Biological Sciences and Technology, Chonnam National University

Dendropanax morbifera is native to southern Korea its sap is used as paint The xylan is a group of hemicelluloses that are found in plant cell wall and of traditional crafting, and now in the spotlight as a material for functional some algae. Xylan are polysaccharides made from units of xylose. So, foods. Recent studies reported Dendropanax morbifera extract shows xylanolytic enzymes from microorganism have attracted a great deal of anti-bacterial, anti-cancer, antioxidant excellent effects. In this study, attention because of their biotechnological characteristics in various industrial Dendropanax morbifera extract is the fermentation using microorganisms processes, related to pulp, food, feed, ethanol, and paper industries. A to make sure they are easier absorption by body and compare its microbial screening of xylanase producer was carried out in this study. antioxidant effects and the end of fermentation ingredients to before About 250 bacterial strains were isolated from soil sample. Among these fermented. 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.

68 | www.msk.or.kr Poster

G037 G039 Dyeing Properties and Antibacterial Activity of Natural Development of Home-made Gibson Assembly Enzymes for Pigment from Marine Bacteria Do It Yourself Biologists

Ga Eun Lee and Jin Sook Park* Jaewon Kim, Saeyoung Lee, Hongjae Park, Kyoungwoo Jang, and In-Geol Choi* Department of Biological Science and Biotechnology, Hannam University Department of Biotechnology, Korea Univeristy

In this study, dyeability on mutifiber fabrics and antibacterial activities Gibson assembly (GA) is a popular cloning method used in the joining of using natural pigment derived from marine bacteria were investigated. multiple DNA fragments in a test tube. Using the commercial kit costs The violet colourant was produced by cultivation of Microbulbifer sp. PPB more than $10 per reaction and still demands good hands in unexperienced 12 using marine broth 2216 for 3 days. The violet pigment dissolved in labs. In order to provide a home-made GA kit for ‘Do It Yourself’ (DIY) 20% ethanol could be dyed on bleached cotton, diacetate, and especially biologists, we sought candidate enzymes for the home-made kit. We polyamid. Optimum temperate, time, pH and bath ratio under dyeing expressed three enzymes under a T7 expression system: a thermophilic 3’ condition were 80–90°C, more than 1 hour, pH 4–6 and 1:25, respectively. to 5’ proofreading DNA polymerase from Pyrococus furiosus (PfuPol), a Mordanting treatment after dyeing 10 minutes was suited to color thermophilic NAD+ dependent DNA ligase from Auqifex pyrophilus (ApyLig) expression, but no significant difference was showed from being not treated. and a DNA exonuclease from T7 bacteriophage (T7Exo). The activity of Also, the violet pigment showed antibacterial activity against Bacillus each enzyme was optimized for various reaction conditions. By mixing subtilis. As a results, the natural colourant from marine bacteria is considered three enzymes in a combinatorial way, we built a home-made GA kit for to be utilized for manufacturing polyamide garment with antibacterial DNA cloning in an isothermal reaction and determined the optimal condition. activity. When we tested with two DNA molecules (200 ng each) overlapped 40 bps [Supported by Ministry of Education (1345231167)] 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.

G038 G040 The Physicochemical Stabilities of Pigment Extracts from RapB Controls Cell Adhesion and Migration in Dictyostelium Erythrobacter sp. PPB2-8 Byeonggyu Park and Taeck J. Jeon* Ga Eun Lee and Jin Sook Park* Department of Life Science & BK21 – Plus Research Team for Bioactive Control Department of Biological Science and Biotechnology, Hannam University Technology, College of Natural Sciences, Chosun University

We investigated the physicochemical stabilities and antibacterial activitiy Ras proteins are small, monomeric GTPases that act as crucial regulators of ethanol- extracted pigment from marine bacterium Erythrobacter sp. of a number of cellular signaling pathways, including proliferation, cell PPB2-8. The bacterial pigment of strain PPB2-8 was very stable at pH 8–9 migration, differentiation and apoptosis. In Dictyostelium genome database, and -20~40°C. In the presence of light, the pigment was also stable, it has been reported that there are 19 Ras subfamily proteins. The showing more than 77% of remaining absorbance during 15 days at 25°C. functions of most of these Ras proteins in development and cell migration The stability of the pigment, when metal ions were present, was higher have not been studied yet. Here we investigated the roles of RapB in cell stability in all examined metal ions except for Cu2+ and Al3+, especially in migration and development. RapB is a Ras subfamily protein showing the the presence of Na+. The result indicates that the bacterial pigments from highest homology (48.9% identity in amino acid sequences) with RapA, marine bacterium, Erythrobacter sp. PPB2-8 showed higher physicochemical which is a key regulator in cell adhesion and cell migration. Similar to stability. Therefore, the results suggest that these bacterial pigments RapA-overexpressing cells, cells expressing constitutively active form of could be used as a natural colorant. RapB displayed flattened and spread morphology and strong cell-substratum [Supported by Ministry of Education (1345231167)] 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]

www.msk.or.kr | 69 2016 International Meeting of the Microbiological Society of Korea

G041 G043 Cytokinesis Defects of rapGAP9 null Cells are Rescued by Adaptive Engineering of a Hyperthermophilic Archaeon on Expressing Truncated GAP-Domain Proteins CO and Discovering the Underlying Mechanism by Multi-omics Analysis ARa Lee and Taeck J. Jeon* 1,2 3 1 1,2 Department of Life Science & BK21-Plus Research Team for Bioactive Control Seong Hyuk Lee , Min-Sik Kim , Jae-Hak Lee , Tae Wan Kim , 1 1 1,2 1 Technology, College of Natural Sciences, Chosun University Seung Seob Bae , Sung-Mok Lee , Hae Chang Jung , Tae-Jun Yang , Ae Ran Choi1, Yong-Jun Cho4, Jung-Hyun Lee1,2, Kae Kyoung Kwon1,2, 1,2 1,2 Rap1 is an important regulator in cell adhesion and cell migration. It has Hyun Sook Lee , and Sung Gyun Kang * 1 2 recently been reported that spatial and temporal regulation of Rap1 Korea Institute of Ocean Science and Technology, Department of Marine 3 activity by RapGAPs is required for development and cell migration in Biotechnology, Korea University of Science and Technology, Korea Institute of 4 Dictyostelium. RapGAP1 is involved in spatiotemporal regulation of cell Energy Research, Chunlab, Inc. adhesion at the front of chemotaxing cells. RapGAP3 is associated with The hyperthermophilic archaeon Thermococcus onnurineus NA1 can grow morphogenesis and development. RapGAP9 is involved in cell adhesion and produce H2 on carbon monoxide (CO) and its H2 production rates have and cytokinesis. Here, we investigated overlapping/specific functions of been improved through metabolic engineering. In this study, we applied RapGAP1, RapGAP3, and RapGAP9 in morphogenesis, adhesion, cytokinesis, adaptive evolution to enhance H2 productivity. After over 150 serial transfers and development by analyzing phenotypes of rapGAP null cells expressing onto CO medium, cell density, CO consumption rate and H2 production each RapGAP protein. Interestingly, almost all phenotypes of rapGAP1, rate increased. The underlying mechanism for those physiological changes rapGAP3, and rapGAP9 null cells in development and cytokinesis were could be explained by using multi-omics approaches including genomic, complemented by expressing any one of the RapGAP proteins (RapGAP1, transcriptomic and epigenomic analyses. A putative transcriptional regulator RapGAP3, and RapGAP9), suggesting that the functions of these RapGAP was newly identified to regulate the expression levels of genes related to proteins are highly overlapped. It has been known that rapGAP9 null cells CO oxidation. Transcriptome analysis revealed significant changes in the are highly multinucleated. These cytokinesis defects of rapGAP9 null cells transcript levels of genes belonging to the categories of transcription, were completely by expressing full-length RapGAP9 or truncated proteins translation and energy metabolism. Our study presents the first genome-scale containing the GAP domain. In contrast rapGAP9 null cells expressing methylation pattern of hyperthermophilic archaea. Adaptive evolution truncated proteins without the GAP domain showed no rescued phenotypes led to highly enhanced H2 productivity at high CO flow rates using synthesis in cell adhesion, cytokinesis and development. These results suggested gas produced from coal gasification. that the GAP domain is essential for the RapGAP9’s functioning in [This work was supported by the KIOST in-house program (PE99413), the Dictyostelium cells. Development of Biohydrogen Production Technology Using the Hyperthermophilic [Supported by Base Science Research Program through NRF] 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.] G042 G044 Proper Regulation of CBP7, a Calcium Binding Protein, is Adaptive Evolution of a Hyperthermophilic Archaeon Required for Development in Dictyostelium Thermococcus onnurineus NA1 during Growth on Formate

Dong-Yeop Shin and Taeck J. Jeon* Hae-Chang Jung1,2, Seong Hyuk Lee1,2, Jung-Hyun Lee1,2, 1,2 1,2 Department of Life Science & BK21-Plus Research Team for Bioactive Control Hyun Sook Lee , and Sung Gyun Kang * Technology, College of Natural Sciences, Chosun University 1Korea Institute of Ocean Science and Technology 2Department of Marine Biotechnology, Korea University of Science and Technology Calcium and calcium binding proteins are necessary for diverse intracellular signaling. In Dictysotelium, fourteen genes encoding calcium binding proteins The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been (CBP) have been found. The functions of most of these CBP proteins in reported to grow on formate coupled with H2 production and ATP generation. development have not been studied yet. CBP7, one of 14 CBPs, is To develop the strain for the enhanced H2 production on formate, composed of 169 amino acids and contains four EF-hand domains. Here, adaptive evolution was adopted by transfering T. onnurineus NA1 to we investigated roles of CBP7 in Dictyostelium development and found formate containing medium. Through serial transfers of batch cultures, that CBP7 appears to play some important roles in the cell aggregation physiological changes were monitored and gradual increases in cell density, step during development. Surprisingly, cells expressing CBP7 showed severe H2 production rate and formate consumption rate were observed. After development defects, complete loss of development, while wild-type cells 156 transfers, the evolved strain showed 1.4-, 1.3- and 1.3-fold higher formed aggregates within 6-8hr development and finally fruiting bodies. values in maximum cell density, H2 production rate and formate consumption CBP7-overexpressing cells did not even aggregate and just stayed at the rate, respectively, in comparison with those of parental strain. In order to single-cell growing stages in the development-inducing condition. Cell sorting obtain an integrative understanding of the genetic and phenotypic changes experiments using mixed cells of wild-type cells and CBP7 overexpressing during evolution on formate, genome sequencing was performed. As a cells confirmed that CBP7-overexpressing cells had much lower migration result, we discovered eleven mutation sites, including 2 insertions, 2 deletions and aggregation speed in the aggregation stage of development compared and 7 substitutions either at the 2 intergenic or 9 coding regions. Further to wild-type cells. The third one of four EF-hand domains in CBP7 appear mutational studies would be required to assess the contribution of gene to be essential for functions of CBP7 in development. Cells expressing mutations to the phenotypic changes during evolution. The potential of CBP7 with mutations in the third EF domain showed normal development. the engineered strains as a H2 producer will also be investigated by kinetic Our results suggest that CBP7 plays an important role in development analysis in a bioreactor. through the third EF-hand domain, possibly calcium binding. [This work was supported by the KIOST in-house program (PE99413) the [Supported by Basic science Research program through NRF] Development of Biohydrogen Production Technology Using the Hyperthermophilic Archaea program of the Ministry of Oceans and Fisheries in the Republic of Korea.]

70 | www.msk.or.kr Poster

G045 G047 DReS: A Direct Cloning Technique Using Bacteriophage Engineering Substrate Selectivity by Active-site Residues in Recombination Systems Ferulic Acid Decarboxylase of Enterobacter sp. Px 6-4 by Site-saturation Mutagenesis Kyoungwoo Jang, Linh Thuy Do, Hongjae Park, and In-Geol Choi* Department of Biotechnology, College of Life Sciences and Biotechnology, Korea Sunil Ghatge, Youri Yang, and Hor-Gil Hur* University School of Environmental Science and Engineering, Gwangju Institute of Science and Technology Molecular cloning in conventional genetic engineering has been dependent on various molecular biology enzymes such as restriction enzymes and Conversion of lignin depolymerization products to value added chemicals ligases. Synthetic biology employs novel cloning techniques such as CPEC, has attracted increasing attention during last few years. Ferulic acid LIC, SLICE or Gibson assembly. Recombineering (recombination-mediated decarboxylase (FADase) catalyzes non-oxidative decarboxylation of lignin genetic engineering) is a synbio cloning method using homologous recom- monomers p-coumaric acid, caffeic acid, and ferulic acid into their bination. This method not only allows successive joining of DNA fragments corresponding 4-vinyl derivatives, which is, 4-vinylphenol, 4-vinylcatechol, having overlaps but also manipulates very large DNA fragments such as and 4-vinylguaiacol, respectively. Among various FADase, we selected the bacterial artificial chromosomes or chromosomes. FADase from Enterobacter sp. Px 6-4, whose crystal structure is known, In order to clone or disrupt a genomic region of bacteria, we developed a and tried to change the substrate selectivity using site saturation and site recombieering method based on bacteriophage recombinases designated directed mutagenesis of active site residues Y27 and F95. Substitution of as DReS (Dual Recombination System) using both λ (Red α/β/γ) and Rac Y27S and F95E of FADase resulted into selective biotransformation of prophage recombination system (RecET). The method is mainly focused p-coumaric acid and ferulic acid in presence of mixture of the three on ‘circular-circular recombination (CCHR)’. Using the CCHR, we attempted substrates. The mutants characterized could be useful for the selective a direct cloning of a large chromosomal region into a plasmid vector or production of valuable chemicals such as 4-vinylphenol and 4-vinylguaiacol. 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)]

www.msk.or.kr | 71 2016 International Meeting of the Microbiological Society of Korea

H001 H003 Antiviral Activity of Bisisomahanin from the Glycosmis Pan-genomic Analysis of Lactobacillus salivarius Strains as stenocarpa Anti-pathogenic Swine Probiotics

Jang Hoon Kim1, Ju Yeon Yoon1, Seung Kook Choi1, Sun Jung Kwon1, Jun-Yeong Lee1, Geon Goo Han1, Gwi-Deuk Jin2, Sang Mok Lee1, In Sook Cho1, Young Ho Kim2, and Gug Seoun Choi1* Yun-Jaie Choi1, and Eun Bae Kim2,3* 1Department of Horticultural Environment, National Institute of Horticultural 1Department of Agricultural Biotechnology, Seoul National University, 2Department and Herbal Science, RDA, 2College of Pharmacy, Chungnam National University of Animal Life Science, College of Animal Life Sciences, Kangwon National University, 3Division of Applied Animal Science, College of Animal Life Sciences, The growth and productivity of the peppers in field and greenhouse were Kangwon National University suppressed by Pepper mild mottle virus (PMMoV). The aim of this study is to search for inhibitor on PMMoV from natural plants. Bisisomahanine Probiotics is a promising candidate for replacement of antibiotics that was isolated from the aerial parts of Glycosmis stenocarpa (G. stenocarpa) were overused in the livestock industry, and in this aspect, antimicrobial by using Silica gel and C-18 column chromatography. It's structure was activity against pathogens is the most important traits of probiotics. In the elucidated by 1H-NMR , 13C-NMR and Mass. This was revealed to have the present study we attempted to identify genetic variety which affect its inhibitory activity through the half-leaf assay against PMMoV. Furthermore, antimicrobial activity for selection of anti-pathogenic probiotics. We first mixture of viral with compound was observed by electron microscope. isolated and identified the probiotic bacteria species, Lactobacillus salivarius This study suggested that bisisomahanine derived from G. stenocarpa that is used in broad field including human and livestock in the swine may be the lead compound for the development of antiviral agents. feces. Antimicrobial activity of isolated L. salivarius strains was measured [This study was supported by the basic research project (PJ010878012015) of against Escherichia coli and Salmonella enterica. L. salivarius strains that Natural Institute of Horticultural and Herbal Science, RDA] 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.]

H002 H004 Marine Fungal Resource Bank Development of Detection Method for Foodborne Pathogens at Low Density on Fresh Produce Jae Young Park, Myung Soo Park, Ji Eun Eom, and Young Woon Lim* Seoul National University Sujin Yun, Jin-Woo Park, Jae-Gee Ryu, and Sanghyun Han* Microbial Safety Team, Department of Agri-Food Safety, National Institute of The Marine Fungal Resource Bank (MFRB), overseen by Dr. Young Woon Agricultural Sciences 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 Foodborne illness outbreaks associated with fresh produce contaminated the MFRB is to establish a culture collection of marine fungi for educational, with human pathogens have resulted in an increasing demand for safe scientific, and industrial purposes. MFRB will undertake following tasks: 1) produce. Under even optimally controlled conditions, fresh produce could Survey marine environments across Korea to catalogue marine fungal be contaminated with fecal matter entailing the potential presence of diversity, 2) Establish a robust system of polyphasic species identification, foodborne pathogens pre- and post-harvest. Population levels of the 3) Evaluate the usefulness of the discovered fungi, 4) Create a secure foodborne pathogens on fresh produce are thought to be very low preservation and loan system, 5) Provide web-based access to the because it is not the host for the pathogens. In this study, in order to database. With a global focus on utilizing natural resources, marine fungal develop a microarray that detects multiple pathogens present on fresh resources provide excellent opportunities for educating the public on produce at very low level, three candidate sets of primers priming conserved marine ecosystem, vitalizing marine research, and discovering novel substances regions within 16S rRNA shared by six different pathogens were tested for for use as medicine and energy. their ability to amplify low concentration of DNA samples. Amplified [This work was supported by the Marine BioResource Bank Program of the regions are variable in size and sequence for each pathogen. All of them Ministry of Ocean & Fisheries.] 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.

72 | www.msk.or.kr Poster

H005 H007 Relationship between the Microbiota in Different Sections Genetic Analyses of Sinonovacula constricta from Southern of the Gastrointestinal Tract, and the Body Weight of Broiler Coast of Korea Based on DNA Sequences of Mitochondrial Cytochrome Oxidase I Gene Geon Goo Han1, Jinyoung Lee2, Jun-Yeong Lee1, Gwideuk Jin3, Jongbin Park4, Changsu Kong2, Eun Bae Kim3, and Yun-Jaie Choi1* Mi Sun Kim, Ji Hee Lee, Joo Won Kang, Dae In Kim, and Chi Nam Seong* 1Department of Agricultural Biotechnology, Seoul National University, 2Department Department of Biology, College of Life Science and Natural Resources, Sunchon of Animal Science and Technology, Konkuk University, 3Department of Animal National University Life Science, Kangwon National University, 4Department of Animal Life System, Kangwon National University Sinonovacula constricta is a benthic clam and one of the important In the poultry industry, many efforts have been undertaken to improve economic shellfish. The mitochondrial cytochrome oxidase subunit 1 (CO the growth performance of broilers and identification and modulation of I) has been widely used in genetic diversity and population genetic structure body weight (BW)-related bacteria could be one of the strategies to improve of marine species. In this study, we compared the partial sequences of the productivity. The aim of the study was to investigate the relationship CO I gene of Sinonovacula constricta. Samples were collected from Goheung between microbiota and BW in different sections of the gastrointestinal and Beolgyo on the southern coast of Korea. Ten haplotypes were identified tract (GIT), and explore the weight related bacterial groups in broiler. A out of 30 individuals. Four haplotypes (A, B, C, D) were recovered from the total of twenty 18-day-old birds were selected based on the BW, and both regions. The most frequent haplotype B consisted of 12 individuals, samples were collected from the 3 different sections of the GIT, which including seven from Goheung and five from Beolgyo. 4 haplotypes (E, F, included the crop, ileum and cecum. Bacterial genomic DNA was extracted G, H) were found only in Beolgyo, but 2 haplotypes (I, J) also occurred in from the samples, and the V4 region of 16S rRNA gene were amplified. Goheung. Amplicons were sequenced on Illumina MiSeq, and microbial communities [This work (Grants No. C0268343) was supported by Business for were analyzed by using QIIME. In principal coordinate analysis, bacterial Academic-industrial Cooperative establishments funded Korea Small and communities were clustered into three groups, based on the sections of Medium Business Administration in 2015.] 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 H008 Prediction and Purification of Sesquiterpene Synthase from Evaluation of Bacterial Community Composition of Wood Rot Fungus Commercial Animal Probiotics from Korea Using Brcoded Pyrosequencing Su-Yeon Lee, Jieun An, and MyungKil Kim* National Institute of Forest Science Baolei Jia, Hyo Jung Lee, and Che Ok Jeon* Department of Life Science, Chung-Ang University Terpenoid natural compounds have been exploited for drugs, fragrance and aroma ingredients. In our previous study, biosynthesis of eudesmane-type A wide variety of probiotics for animal feeding have been used because sesquiterpenoids which are composed with three isoprene unit, have the use of antibiotics for animal as growth promoters was prohibited. been identified from wood rot fungus, Polyporus brumalis. In this study, However, just few studies have been conducted to evaluate the microbial transcriptome analysis has been conducted to understanding the terpenoid compositions for commercial probiotic products, whether labeling on the metabolism in P. brumalis. RNA sequencing of P. brumalis was performed products for animal feeding represents the true microbiological contents using PacBio next generation sequencing suing Single Molecule Real Time. or not. Bacterial community compositions of fifty commercial probiotics of Non-redundant transcripts (11,143) obtained by isoforms statistics analysis twenty-six brands in Korea were investigated using a barcoded pyrosequencing were annotated as BLASTX result against GO database. Three sesquiterpene approach. Pyrosequencing data were processed and classified using synthases has been predicted from three wood rot fungi, Gloephyllum RDPipeline and the presence of potential pathogenic bacteria were estimated trabeum, Dichomitus squalenes and Trametes versicolor. Differentially by Blastn based on PHI (Pathogen Host Interaction) database. Labeling on expressed genes (DEGs) analysis of annotated genes has been performed the products showed that Lactobacillus, Bacillus, Clostridium, and Saccharomyces in the three mycelium which were inoculated in cultures of control, magnesium were major components. The pyrosequencing data indicated that some blank and phosphate blank. As the results, the highest expression level of probiotics were relatively well accordance between bacterial claims on G. trabeum sesquiterpene synthase was shown in phosphate blank culture. their labels and classification results. However, some differences were Also, the yield of eudesmane type sesquiterpenoids was obtained from also observed in some probiotic products; some probiotic products even phosphate blank culture. To investigate a gene function of predicted contained not declared bacteria. About 2% of the total sequences were sesquiterpene synthases on terpene metabolism of P. brumalis, purification closely related to potentially pathogenic sequences such as Pseudomonas and characterization of sesquiterpene synthase have being researched. 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.]

www.msk.or.kr | 73 2016 International Meeting of the Microbiological Society of Korea

H009 H011 A Phylogenic, Functional and Metabolic Analysis of Bacillus Antimicrobial Activity of Essential Oil of Eucalyptus globulus velezensis Using Pan Genome against Fish Pathogenic Bacteria

Byung Hee Chun and Che Ok Jeon* Joon-Woo Park, Mitchell Wendt, Sabrina Hossain, Department of Life Science, Chung-Ang University Sudu Hakuruge Madusha Pramud Wimalasena, and Gang-Joon Heo* College of Veterinary Medicine, Chungbuk National University Bacillus velezensis is a rod-shaped, spore-forming and Gram-positive bacterium that grows under strictly aerobic conditions. In this study we The antibacterial activities of the essential oil of Eucalyptus globulus performed the pan and core genome analysis of B. velezensis to investigate (EOEG) was determined against 7 fish pathogenic bacteria (Edwardsiella tarda, phylogenic, functional and metabolic features of B. velezensis. We retrieved Streptococcus iniae, S. parauberis, Lactococcus garviae, Vibrio harveyi, V. all genomes (46) of B. velezensis and its relatives (named as B. velezensis, ichthyoenteri and Photobacterium damselae) obtained from farmed olive B. amyloliquefaciens, B. methylotrophicus, and B. siamensis in GenBank) flounder. The inhibitory activity was evaluated by three methods: Disc from GenBank and checked their qualities using CheckM. Average nucleotide diffusion method, minimum inhibitory concentration (MIC) and minimum identity analysis of eventually obtained 46 genomes showed that 40, 4, bactericidal concentration (MBC). According to the disc diffusion test, as and 2 genomes were classified into B. velezensis, B. amyloliquefaciens, the concentration of EOEG (5-40 μg) rises, the inhibitory zone increases in and B. siamensis, respectively and a phylogeny tree using 2054 core genes size. Compared with amoxicillin, tetracycline and chloramphenicol, EOEG also supported that they were clearly split into different phylogenetic showed similar antibacterial activity. The MIC of EOEG ranged from 7.8 to lineages–– a phylogenetic analysis based on 16S rRNA gene sequences did 125 mg/ml and MBC values ranged from 62 to 250 mg/ml. These results not clearly differentiate them. The genome analysis of forty B. velezensis show that EOEG has antimicrobial activity against all seven bacteria, but strains was found to contain 5932 pan genes and 2490 core genes. COG there was no marked difference between each genus. From these results, distribution analysis using core genome revealed that B. velezensis contains it is suggested that EOEG can be used as an antimicrobial agent against high fractions for amino acid transport and metabolism and transport fish bacterial diseases in the fish industry. categories and B. velezensis harbors various antimicrobial activity and salt tolerance-related genes.

H010 H012 Pan-genome Analysis of Leuconostoc mesenterides Reveals Morphological Changes of MG-63 Cells by Fucoidan Treatment Its Metabolic Capabilities and Diversities during Fermentation Hyeseon Kim and Taeck J. Jeon* Hye Hee Jeon and Che Ok Jeon* Department of Life Science & BK21-Plus Research Team for Bioactive Control Department of Life Science, Chung-Ang University Technology, College of Natural Sciences, Chosun University

To investigate the comprehensive characteristics of Leu. mesenteroides, Fucoidan, a fucose-rich sulfated polysaccharide which is found in brown we performed the pan-genome analysis using whole genome sequences algae, possesses diverse biological activities including antibacterial, antioxidant, of sixteen Leuconostoc (Leu.) mesenteroides strains available in GenBank anticoagulant, anti-inflammatory, and antitumor activities. Recently, the database–– average nucleotide identity values also supported that all they fucoidan has been reported to induce apoptosis in various cell lines. In this belong to Leu. mesenteroides, The pan-genome of the sixteen Leu. study, the apoptotic effects of fucoidan were investigated by analyzing mesenteroides strains was found to contain 3088 genes and 716 core morphological changes in MG-63 osteosarcoma cells in the presence of genes. A phylogenetic tree was constructed using all core genes to investigate fucoidan. We founded that the viability and proliferation of MG-63 cells their phylogenetic relationships, which were a little different from the was decreased in the presence of fucoidan in a dose- and time-dependent phylogenetic relationships based on 16S rRNA gene or house-keeping manner. Fucoidan-treated cells became shrunk and rounded. The cells gene sequences and they could be grouped into four subspecies. For the were highly aggregated and much smaller than control cells. To further analysis of functional capabilities of Leu. mesenteroides, all protein sequences understand these morphological changes of the cells in the presence of were assigned into respective COG categories. The COG analysis showed fucoidan, we examined the fragmentation of DNA and apoptotic effects of that Leu. mesenteroides species have high fraction for transport and fucoidan. When the cells were treated with fucoidan, some fragmented metabolism of carbohydrates and amino acids, which is similar with other DNAs were observed while there was no such fragmented DNA in the Leuconostoc species. In addition, to investigate metabolic capability of absence of fucoidan. In addition, significantly increased number of cells Leu. mesenteroides, all functional genes of sixteen genomes were mapped was stained by AnnexinV in the presence of fucoidan, compared to control onto the KEGG pathways. As the results, genes associated with carbohydrate cells. Our results suggest that fucoidan has apoptotic effects on MG-63 and amino acid metabolism and PTS systems related to sugar transports cells with morphological changes. were abundant, suggesting that Leu. mesenteroides was evolved to adapt [This research was surpported by Basic Science Research Program through to environments with various carbon sources. This is the first study to NRF.] investigate the metabolic properties of Leu. mesenteroides.

74 | www.msk.or.kr Poster

H013 H015 Effectiveness of Periodic Treatment of Quercetin against Anti-Norovirus Activity from Natural Plant Extract Using a Viral Hemorrhagic Septicemia Virus Norovirus Surrogate System

Se-Young Cho1, Yeong O Kim2, Se Hyun Cho2, Jong-Soon Choi3, Joo Bong Choi1, Diana Soils Sanchez1, Hee Jung Lee2, In Sun Joo2, Joseph Kwon3, and Duwoon Kim1,2* Jeong Su Lee2, and Sung-Joon Lee1* 1Foodborne Virus Research Center, Chonnam National University 1Department of Biotechnology, College of Life Sciences and Biotechnology, 2Department of Food Science and Technology, Chonnam National University Korea University 3Korea Basic Science Institute 2Food Microbiology Division, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation Viral hemorrhagic septicemia virus (VHSV), a rhabdoviral fish pathogen, creates a major threat to the development of aquaculture industry worldwide. Norovirus (NoV) has been identified as a major cause of acute gastroenteritis The fathead minnow (FHM) cells infected with VHSV at a multiplicity of epidemics worldwide. This study investigated the effect of Extract A on infection (MOI) of 0.2 and treated with 100 μl quercetin. The quercetin the infectivity and viral replication of norovirus. Murine norovirus (MNV), treatment showed cell viability of 64.3% at 42 h post infection, while a surrogate of human norovirus, was pre-incubated at titers of ~5 log10 non-treated cells infected with VHSV at the same MOI had a cell viability of PFU/ml with Extract A and then used to infect RAW 264.7 cells in a plaque 23.7%. The highest cell viability was found by the treatment of quercetin reduction assay. MNV incubated with Extract A at 4°C exhibited a significant (Q) and followed by kaempferol (K), taxifolin (T), catechin (C), myricetin reduction in MNV plaque formation in both time- and dose-dependent (M), quercetin 3-O-α-L-arabinoside (Q3A), quercetin 3-O-rutinoside (Q3R), manners. qPCR results revealed that the viral RNA in the RAW 264.7 host quercetin 3-β-D-glucuronide (Q3Glu), quercetin 3-β-glucoside (Q3G), cells infected with Extract A-treated NMV was significantly reduced compared kaempferol 3-O-β-D-glucoside (K3G) and quercetin 3-β-D-galactoside to that in cells infected with untreated MNV. These results suggest that (Q3Gal). In this study, the effect of pretreatment and periodic treatment Extract A inactivates norovirus and its subsequent replication in host cells. with quercetin against VHSV in FHM cells. Compared to pretreatment, Thus, Extract A shows promise as method of inhibiting norovirus within periodic treatment resulted in significantly higher cell viability but lower the food industry. relative expression of the VHSV titration and total apoptosis and cell [Supported by grants from KFDA] 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 H016 A Functional Annotation System Based on Functionally The Role of Sir2 in Oxidative Stress Response Changes Equivalent Protein/Domain Search Algorithm Depending on the cAMP-PKA Signaling

Dong Su Yu Yeong Hyeock Kim, Woo Sun Song, Woo Kyu Kang, and Jeong Yoon Kim* National Institute of Ecology Department of Microbiology and Molecular Biology, College of Bioscience and Biotechnology, Chungnam National University In a functional annotation system, homology-based transfer (HBT) is a gold standard for transferring the functional description of homologs to Silent Information Regulator 2 (Sir2), which is a NAD+-dependent protein the putative proteins. To search homologs for the putative proteins, deacetylase, is known to contribute to calorie restriction (CR)-mediated sequence-based methods such as BLAST and HMMER have been commonly lifespan extension in yeast. However, the link between Sir2 and oxidative used because of fast homolog searches against big databases. Although stress in CR-mediated lifespan extension remains elusive. We have recently many putative proteins have been annotated by HBT using sequence-based reported that the role of Sir2 in oxidative stress resistance and chronological methods with high confidence, meticulous attention is still required owing lifespan is dependent on growth phase in yeast. In this study, we show to the emergence of misannotated or unannotated proteins caused by that the cAMP-PKA signaling determines the Sir2’s role in oxidative stress missing functionally equivalent homologs. Actually, the performance of response. Deletion of PDE2, which increases cAMP level, changed the HBT-based annotation system using sequence-based methods is considerably negative role of Sir2 for H2O2 resistance to the positive during early dependent upon the ability of detecting functionally equivalent homologs. exponential phase. In contrast, deletion of RAS2, which reduces cAMP We developed an HBT program based on functionally equivalent proteins level, reversed the positive role of Sir2 for H2O2 resistance to the negative (FEPs) or domains (FEDs) using the FEP-BH algorithm, called HBT-FEPD. As during post diauxic phase. Additionally, buffering of culture medium to pH FEP-BH algorithm is used to detect FEPs from the outputs of blastp and 6.0, which is known to inactivate the Ras/cAMP-PKA pathway, made sir2 hmmsearch, HBT-FEPD can more precisely annotate the putative proteins mutant cells more resistant to H2O2 than wild type during post-diauxic by the selected FEPs and FEDs. 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.

www.msk.or.kr | 75 2016 International Meeting of the Microbiological Society of Korea

H017 H019 Immunogenicity and Efficacy of VLP Forming Baculoviral Chronic Repression of mTOR Complex 2 Alters the Composition Vaccine against Influenza pdmH1N1 in BALB/c Mice of the Gut Microbiota in Diet-induced Obese Mice

Yong-Dae Gwon, Sehyun Kim, Yoonki Heo, Hansam Cho, Yeondong Cho, Mi-Ja Jung, Jina Lee, Min-Soo Kim, Dong-Wook Hyun, Na-Ri Shin, Ki Hoon Park, Yuyeon Jang, Jong Kwang Yoon, Ji-Hyun Yun, Pil Soo Kim, Tae Woong Whon, and Jin-Woo Bae* Hee-Jung Lee, and Young Bong Kim* Department of Life and Nanopharmaceutical Sciences and Department of Department of Bio-industrial Technologies, Konkuk University Biology, Kyung Hee University

An outbreak of influenza H1N1 in 2009, representing the first influenza Alterations in the gut microbiota play a crucial role in host physiology and pandemic of the 21st century, was transmitted to over a million individuals metabolism; however, the molecular pathways underlying these changes and claimed 18,449 lives. The current status in many countries is to in diet-induced obesity are unclear. Mechanistic target of rapamycin prepare influenza vaccine using cell-based or egg-based killed vaccine. (mTOR) signaling pathway is associated with metabolic disorders such as However, traditional influenza vaccine platforms have several limitations. obesity and type 2 diabetes (T2D). Therefore, we examined whether To overcome these limitations, many researchers have tried various approaches changes in the regulation of mTOR signaling induced by diet (a high-fat to develop alternative production platforms. One of the alternative approach, diet [HFD] or normal-chow diet) and/or therapeutics (resveratrol [a specific we constructed a human endogenous retrovirus (HERV) envelope-coated, inhibitor of mTOR complex 1] or rapamycin [an inhibitor of both mTOR baculovirus-based, virus-like-particle (VLP)–forming DNA vaccine (termed complex 1 and 2]) altered the composition of the gut microbiota in mice. AcHERV-VLP) against pandemic influenza A/California/04/2009 (pH1N1). Oral administration of resveratrol prevented glucose intolerance and fat BALB/c mice immunized with AcHERV-VLP (1×107 FFU AcHERV-VLP, i.m.) accumulation in HFD-fed mice, whereas rapamycin significantly impaired and compared with mice immunized with the killed vaccine or mice glucose tolerance and beta cell function. The abundance of Lactococcus, immunized with AcHERV-HA. As a result, AcHERV-VLP immunization produced Clostridium XI and Hydrogenoanaerobacterium increased under HFD condition; a greater humoral immune response and exhibited neutralizing activity however, the abundance of these species declined after resveratrol with an intrasubgroup H1 strain (PR8), elicited neutralizing antibody production, a treatment. Conversely, the abundance of unclassified Marinilabiliaceae high level of interferon-γ secretion in splenocytes, and diminished virus and Turicibacter decreased in response to a HFD or rapamycin. Taken shedding in the lung after challenge with a lethal dose of influenza virus. In together, these results demonstrated that changes in the composition of conclusion, VLP-forming baculovirus DNA vaccine could be a potential intestinal microbiota, resulted from differential mTOR activity, correlated vaccine candidate capable of efficiently delivering DNA to the vaccinee with obese and diabetic phenotypes, suggesting mTOR pathway as a novel and VLP forming DNA eliciting stronger immunogenicity than killed vaccines. therapeutic target for obesity and T2D. [Supported by grants from iPET] [Supported by grants from NRF and MAFRA.]

H018 H020 Enhanced Immune Response for Foot-and-Mouth Disease Effects of Freeze-drying Feces on 16S rRNA Based Microbial Virus Vaccine with Granulocyte Macrophage Colony Community Analysis Stimulating Factor-flagellin Adjuvant Jungman Kim, Nakwon Hwang, Mincheal Kim, Yu Yeon Jang Jang1, Hansam Cho1, Yong-Dae Gwon1, Hanul Choi1, Son G. Nguyen, and Tatsuya Unno* 1 1 2 Jaehyuck Heo , Gwonsung Joo , Joong-Bok Lee , Faculty of Biotechnology, College of Applied Life Science, SARI, Jeju National 1 1 Jiwon Choi , and Young Bong Kim * University 1Department of Bio-industrial Technologies, Konkuk University, 2Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University Since next generation sequencing (NGS) was developed, microbial communities in intestine and environments have been widely investigated. Especially, Foot-and-mouth disease (FMD) is a highly infectious and economically gut microbiota studies have been published number of times. These devastating disease of cloven-hoofed animals. Here, this study was utilized studies have suggested that gut microbiota was related to intestinal diseases Granulocyte-macrophage colony-stimulating factor (GmCSF) and Salmonella such as obesity, diabetes, diarrhea and cancer. While these results were typhimurium Flagellin 2 (STF2) as adjuvant. GmCSF-STF2 fusion was obtained from DNA sequences obtained from fecal samples, previous studies constructed a recombinant baculovirus, which is connected 2A-linker have reported that results could be varied based on DNA extraction peptides and encoding adjuvant (Ac-ie1-PERV-GmCSF-STF2). GmCSF and methods, DNA concentrations used in PCR, PCR conditions, and sample STF2 genes were confirmed gene expression levels by reverse transcription homogenization. Freezing drying fecal samples prior to DNA extraction is PCR in PK15 cells. NF-κB (nuclear factor kappa-light-chain-enhancer of an ideal method for effective DNA extraction, consistent fecal amount, activated B cells), which is induced by Toll-like-receptor 5 (TLR5) signaling sample homogeneity, and long term storage. In this study, we examined if pathway, was detected by reverse transcription PCR in PAM cells. To freeze-drying fecal samples affect sequence-based microbial community investigate the effects as adjuvant, immunized with commercial FMD analysis by comparing microbial communities of fresh and freeze-dried vaccine in mice. The immunological effects of the Ac-ie1-PERV-GmCSF- fecal samples. Our results showed that number of rare operational taxonomic STF2 were determined specific FMDV by ELISA, ELISPOT, and CTL assay. units (OTUs) was 1.2–1.5 times lower in freeze-dried feces than that of GmCSF-STF2 fusion showed higher IgG titer, INF-γ secretion, and CTL fresh feces while no significant differences were observed between overall assay than that of FMD vaccine only. Extending these result, GmCSF-STF2 community analysis. Substantial rare OTUs are known artificial errors in could stimulate both humoral and cell-mediated immune response as an MiSeq, thus reduced number of rare OTUs indicate better sequencing adjuvant for FMD vaccine. In conclusion, STF2 and GmCSF could be useful quality, suggesting that freeze-drying feces may not only provide benefits for potential vaccine adjuvant against pathogen. such as long-term storage and better homogenization, but also increases [This research was supported by Fishery by iPET (Korea Institute of quality of MiSeq-based microbiota analysis results. Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries) from Ministry of Agriculture.]

76 | www.msk.or.kr Poster

H021 H023 The Roles of Histone H3-K4 Methylation in Morphogenesis PAF1 Complex Directly Regulates H3K4 Methylation in of Candida albicans Saccharomyces cerevisiae

Jueun Kim and Jung-Shin Lee* Jun-Soo Oh and Jung-Shin Lee* Department of Molecular Bioscience, College of Biomedical Science, Kangwon Molecualr Biochemistry Lab, Department of Molecular Bioscience, College of National University Biomedical Science, Kangwon National University

Candida albicans is the most common fungal pathogen in human and its In eukaryotes, many aminoacid residues of histone components can be ability to grow as a filamentous form is important for its virulence. In easily modified by many histone modifying enzymes and this histone hyphal formation of C. albicans, the transcriptional activation of hypha-specific modifications are very important in transcriptional regulation. H3 Lys4 genes is very important. The methylation of histone H3 at lysine 4 (H3K4) methylation can be conducted in mono, di, and tri. Monoubiquitination in is an epigenetic mark studied extensively because it is related to transcription H2B at K120(K123 in yeast) is prerequisite for di- and tri-methylation of by RNA polⅡ. Here, we observed that the Δset1 strain, a deletion mutant H3K4. In other words, histone crosstalk between this two modifications exist. of H3K4 methyltransferase gene, develops hyphae more rapidly. To identify It is reported that PAF1 complex contributes to the H2B monoubiquitination. In the relationship between H3K4 methylation and the transcription of yeast, PAF1 complex consists of PAF1, LEO1, CDC73, CTR9 and RTF1. This hypha-specific genes in C. albicans, we performed the RNA-seq of Δset1 complex is structurally, functionally well conserved from yeast to human. strain in normal condition or hyphal induced condition. The expression It is already reported that PAF1 complex is important in regulating H2B levels of hypha-specific genes in Δset1 strain were more increased than monoubiquitination and indirectly regulates the H3K4 di- and tri-me- those of WT. To investigate the precise role of H3K4 methylation in C. thylation by histone crosstalk. But, we suggests that PAF1 complex directly albicans morphogenesis, we analyzed the localization of each H3K4 regulates the H3K4 methylation, in addition to the reported indirect manner. methylation status in each condition. Surprisingly, we found that the [Supported by grants from NRF-2015R1D1A1A02061743 and NRF-2015- H3K4me2 and me3, generally localized at active transcribed genes, is not R1A4A1041105] 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 H024 Histone Residues Play Important Roles for HM Silencing Korea National Microorganisms Research Resource Center Maintenance in Saccharomyces cerevisiae Se Joung Yeom and Sang Seob Lee* Soojin Yeom and Jung-Shin Lee* Kyonggi University Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University The Korea National Microbiological Research Resource Center is the core center of the twelve microorganism banks designated by the Ministry of Gene silencing is one of important concepts for the epigenetic gene Education, Science and Technology. The KNMRRC supports microorganism regulation. Histone modifications are critical factors for the maintenance banks with necessary guidelines, standards, training for efficient operation of gene silencing in eukaryotic systems. However, none of histone modifications of the banks. It also provides with an effective forum to solve common as epigenetic silencing markers is conserved in Saccharomyces cerevisiae. issues of the related banks. To identify novel global epigenetic silencing marker from yeast to human, The ultimate goal of the KNMRRC is the followings: 1-construction of we developed screening method using the system for the maintenance of standardized and integrated management system, 2-construction of Core yeast mating type. We used yeast histone library, which is a collection of center and other organs network, 3-Quality Control (QC) of microbial resources strains containing alanine-substituted histone residue, and yeast single-gene in the member banks, 4-conservation of Resources in the member banks knockout library. From this screeing, we found that the 79th lysine of and the interrupted banks, 5-education for professionals in the member histone H3 (H3K79) is required for the maintenance HM silencing, but banks, 6 public Relations for raising people's awareness of the importance Dot1, methyltransferase of hisotne H3K79 is not. Also we checked several of microbiological resources. histone residues are required for the maintenance of HM silencing in S. cerevisiae. [This work is supported by NRF-2015R1D1A1A02061743 and NRF-2015- R1A4A1041105]

www.msk.or.kr | 77 2016 International Meeting of the Microbiological Society of Korea

H025 H027 Center for Fungal Genetic Resources (CFGR): Housing Plant Microbial Carbohydrate Resource Bank (MCRB) Pathogenic Fungi for Educational and Research Purposes Seunho Jung Yeo Kyoung Yoon and Yong-Hwan Lee* Department of Bioscience and Biotechnology, Microbial Carbohydrate Resource Center for Fungal Genetic Resources, Seoul National University Bank(MCRB) & Center for Biotechnology Research in UBITA (CBRU), Konkuk 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 Microorganisms have played important roles in biotechnology and bioindustry in the decomposition of organic matter. They have been used as a source for long times. The recent use of molecular ecological methods and of food, and agents for fermentation of food products and for the production environmental DNA (eDNA) has changed our knowledge of microbial of various antibiotics and enzymes that are used in a field of research, diversity dramatically and provided rapid access to genes of yet-uncultured industry, medicine, etc. In contrary, impact of many fungi on animals and microorganisms. Application of molecular ecological studies has shown plants is economically and socially detrimental. For example, Magnaporthe that the majority (99%) of microorganisms present in the nature are under oryzae causes the most destructive disease, “rice blast”. Annual yield loss uncultivation. Many attempts to improve the recovery of microorganisms of rice by rice blast is equivalent to rice that could feed about 60 million and their genes from the environmental samples have recently been people. The Center for Fungal Genetic Resources (CFGR) was established achieved. Metagenomic approach that recovers the environmental DNA to collect, maintain and distribute genetic resources mainly from plant without the limitations of culture-dependent methods and constructs pathogenic fungi, which are important for both educational and research DNA libraries in suitable cloning vectors and host strains have been purposes. This will contribute to development of new strategies for utilized for retrieving novel and useful genes. Korean Metagenome Bank management of crop diseases and of new components for improvement (KMGB), a member of Korea National Research Resource center (KNRRC), of our lives. CFGR possesses important fungal species; a total of 42,000 has opened with the goal for the collection and distribution of metagenome isolates from 54 species of fungi including 20,902 T-DNA transformants of (eDNA) and metagenomic library. The aims of the Korean Metagenome rice blast fungus and anthracnose fungus. In addition to the biological Bank are to contribute to the development of biotechnology by providing materials, CFGR has developed user-friendly databases to maintain genetic the metagenomic resources into various researches and to perform a information of fungal stocks and help to solve questions about fungal national mission for maintaining the metagenomes as future biological pathogenicity, population genetics, development, and evolution. Also, resources CFGR seeks strategies for sustainable and scientific plant quarantine to Keywords: Metagenome, library, environmental DNA better protect our ecosystem from invasive microorganisms. Keywords: Plant pathogen, Fungi, Mutant, Plant quarantine, Pathogenicity genes, Genetic resource

H026 H028 Korean Metagenome Bank for Exploiting Microbial Diversity Bacteriophagebank

Jung-Hoon Yoon KyoungEun Cha and Heejoon Myung* Department of Food Science and Biotechnology, Sungkyunkwan University Dept. of Bioscience and Biotechnology, Hankuk University of Foreign Studies

Microorganisms have played important roles in biotechnology and bioindustry Bacteriophages are viruses growing on bacterial hosts. They are antagonistic for long times. The recent use of molecular ecological methods and to bacteria and first reported by Frederick Twort and Felix d'Herelle in environmental DNA (eDNA) has changed our knowledge of microbial 1915 and 1917, respectively. They are found in sea, air, land and even diversity dramatically and provided rapid access to genes of yet-uncultured foods. It is assumed that 1030 to 1032 phages exist on earth and they play a microorganisms. Application of molecular ecological studies has shown role in maintenance of biological balance. Recently, new applications for that the majority (99%) of microorganisms present in the nature are under phages are increasingly reported. As they are a part of useful biological uncultivation. Many attempts to improve the recovery of microorganisms resources, there are increasing demands for securing these resources. In and their genes from the environmental samples have recently been response to these demands, the bacteriophage bank was established in achieved. Metagenomic approach that recovers the environmental DNA 2010. The bank collects phages from environments as well as from working without the limitations of culture-dependent methods and constructs groups worldwide. Currently, 600 different phages are stocked. The host DNA libraries in suitable cloning vectors and host strains have been utilized bacteria include E. coli, Salmonella enteritidis, Pseudomonas aeruginosa, for retrieving novel and useful genes. Korean Metagenome Bank (KMGB), Listeria monocytogenes, Acinetobacter, Camphylobacter jejuni, Enterococcus a member of Korea National Research Resource center (KNRRC), has faecium, Enterococcus faecalis, Cronobacter sakazakii, Seratia marcescens opened with the goal for the collection and distribution of metagenome and Staphylococcus aureus. The number of stock is growing continuously. (eDNA) and metagenomic library. The aims of the Korean Metagenome The bank also serves as a distributor for the collected phages. (www. Bank are to contribute to the development of biotechnology by providing phagebank.or.kr) the metagenomic resources into various researches and to perform a Keywords: Bacteriophage, 600 different phages, useful biological resources national mission for maintaining the metagenomes as future biological resources Keywords: Metagenome, library, environmental DNA

78 | www.msk.or.kr Poster

H029 H031 Korea Mushroom Resource Bank Plant Virus GenBank Ki Hyun Ryu Jae Young Park, Nam Kyu Kim, Hey Young Choi, Mi Jin So, and Young Woon Lim* Dept. of Horticulture, Biotechnology and Landscape Architecture Seoul National University Plant Virus GenBank (PVGB) is a nonprofit semi-governmental organization, The Korea Mushroom Resource Bank (KMRB) was launched as a national one of the Korea National Research Resources Collections (KNRRC) for research resource bank in 2015 by the Ministry of Science, ICT and Future special research materials Banks program financially supported by the Planning. The main goal of the KMRB is to secure important biological Ministry of Education, Science & Technology (MEST) dedicated to collection, resources, mushroom-forming , significant sources of fun- identification, characterization, preservation, research development, distribution damental and novel substances and materials, as dried specimen, cultures, and deposition of plant virus research biomaterials established since and genomic DNA. For wider application of fungal resources in education, 1999. PVGB is one of substructure of Korea National Microbiological medicinal and industrial uses, the KMRB will undertake following tasks: 1) Research Resources Collections (KNMRRC). PVGB retains a number of Survey natural environments across Korea to catalogue mushroom diversity, accessions and a wide range of collections of Plant Virus Biomaterials 2) Establish resource management system based on accurate identification useful for Plant Virology and Biotech-related research areas. PVGB has of mushroom, 3) Evaluate the usefulness of the discovered mushroom, 4) moved to its current status on November in 2000 and has modern Create a secure preservation and loan system. With a global focus on utilizing facilities and infrastructures for supporting broad research fields as well as natural resources, mushroom resources provide excellent opportunities Plant Virology Community. PVGB has been recognized as a member of for academic research, and discovering novel substances for use as medicine World Federation for Culture Collection & World Data Center for Microorganisms and energy. (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 Keywords: Korea Mushroom Resource Bank, Mushroom, Fungi, Culture, follow ; collection and development of Plant Virus Research Biomaterials Collection 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. H030 H032 Korea Bank for Pathogenic Viruses Lichen as a Novel Bioresources in Korea

Ki-Joon Song Young Jin Koh and Jae-Seoun Hur* Korea Bank for Pathogenic Viruses Korean Lichen Research Institute, Sunchon National University

Korea Bank for Pathogenic Viruses (KBPV) has been established in 2005 as Lichens are symbiotic organisms composed of a fungus (mycobiont) and a repository agent for the collection, management and distribution of the an alga (photobiont). They produce characteristic secondary metabolites, various pathogenic viruses that are essential to use for researches in lichen substances, which seldom occur in other organisms. Lichen and biomedical sciences. The Institution operates in collaboration with The their metabolites have many biological activities. In spite of the wide Institute for Viral Disease at Department of Microbiology, College of Medicine, spectrum of biological activities shown by the lichens, they have long been Korea University, founded in 1973. neglected by mycologists and overlooked by agrochemical industry because The bank has unique viral collections such as Hantaan, Seoul, Muju, of its slow growth in nature and difficulties in the artificial cultivation of Soochong, and imjin the etiologic agents of hemorrhagic fever with renal organisms. Use of lichen-forming fungi can overcome the disadvantage of syndrome. To date, total of more than 43,000 materials (~100,000vials) natural lichen extracts for industrialization of their metabolites because of from human and animal have been collected and maintained. their much faster growth and larger production of the metabolites in culture We have provided a highly collaborative environment for researchers in than the natural thalli. Korean Lichen and Allied Bioresources Center various fields by providing valuable viral resources including consulting focuses on isolation, maintenance and distribution of lichen bioresources service. We also provide the educational program related to pathogenic to research groups in universities, national institutes and industrial sectors. viruses including biosafety training. It also screens their biological activities, and investigates cultural conditions Requestors of such agents are required to register with KBPV and to for large production of lichen substances. Chemical library of some lichen supply details of their laboratory facilities and safety management. More extracts is also available from the center. details about KBPV can be found at ; http://kbpv.knrrc.or.kr Keywords: lichen, lichen-forming fungi, photobiont Keywords: pathogenic viruses, biosafety, viral disease, genetic information, antibody, biomaterial, vaccine, epidemiology, public health

www.msk.or.kr | 79 2016 International Meeting of the Microbiological Society of Korea

H033 H035 Korean Collection for Oral Microbiology Korea Environmental Microorganisms Bank

Soon-Nang Park, Yun Kong Lim, Eojin Jo, and Joong-Ki Kook* Yong Jin Kim and Sang Seob Lee* Department of Oral Biochemisty, School of Dentistry, Chosun University Research Center Kyonggi University

It has been known that about 700 species of oral bacteria inhabit the Korea Environmental Microorganisms Bank (KEMB) has been established human oral cavity. Of them, 350 species have been cultured. The oral as a microbial and genetic resource center for environmental industries. bacteria are the major causative agents of systemic diseases such as The KEMB plays an essential role as follows: 1-the collection and conservation of cardiovascular diseases as well as oral diseases, periodontitis and dental native environmental microorganisms and genetic resources, 2-the construction caries. However, the causative bacterial species for oral diseases have not of systematic management system for effective conservation and application been known because the dental diseases are occurred by the multiple of microbiological resources for environmental industries, 3-the provision infections. In addition, the prevalence of the oral bacterial species is fundamental data for ecosystem research and microbial classification, different by the geographic location of the host and individual. It is very and 4-the development of biological treatment system for bioremedation important to obtain the oral bacteria from Koreans for pathogenesis studies of environmental pollutant and ecosystem restoration. related to oral infectious diseases. The purpose of Korean Collection for There are about 14,000 strains of bacteria collected from environments, Oral Microbiology is to obtain the oral clinical strains and their genetic at this time. These collections are classified in accordance with scientific resources, such as 16S rDNA, species-specific PCR or qRT-PCR primers, and functional characteristics, respectively. and genome sequences, for offering them to the researchers. 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

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

80 | www.msk.or.kr