Dissertation for Degree of Doctor Isolation Of Unculturable Soil Bacteria And Finding Of Their Antibiotic Candidates by Nguyen Manh Tuan Department of Life Science Graduate School Kyonggi University (2017) Isolation Of Unculturable Soil Bacteria And Finding Of Their Antibiotic Candidates by Nguyen Manh Tuan A dissertation submitted to the faculty of the Graduate School in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Life Science Graduate School Kyonggi University December 2017 Approved the dissertation of Nguyen Manh Tuan as qualified for the Degree of Doctor of Philosophy Chairman Signature Member Signature Member Signature Member Signature Member Signature December 2017 Graduate School of Kyonggi University Contents List of Tables ………………………………………………………………………… vii List of Figures ………………………………………………………………………… viii Acknowledgement x Abstract xi Chapter I Modification Of Soil Extraction Method To Cultuvate Unculturable Soil Bacteria 1.1 Introduction 1.2 Aims to Study 1.3 Material and method 1.3.1 Information of soil property used for the method 1.3.2 Preparing new soil extraction (NSE) in litre medium 1.3.3 Medium for isolation of soil bacteria 1.3.4 Soil sampling site and preparation of soil sample 1.3.5 New developed method for isolation of previously unculturable soil bacteria 1.3.6 Traditional soil extract method 1.3.7 Modified transwell culture system method 1.3.8 Bacterial genomic extraction and small-subunit rRNA (SSU rRNA) amplification 1.3.9 Identify 16S rRNA gene sequences and accession numbers of 16S rRNA gene sequenses -i- 1.3.10 Parameter tools/software programs 1.3.11 Role of complex isolate medium compositions to grow previously unculturable soil bacteria 1.3.12 DNA extract from soil 1.3.13 PCR amplification and pyrosequencing 10 1.3.14 Analysis of pyrosequencing data 10 1.3.15 Determining and comparison of soil extract ingredients investigated 11 1.3.16 Sample preparation for simultaneous profiling analysis of amino acids, organic acids and fatty acids in soil extract 12 1.3.17 Gas chromatography-mass spectrometry 12 1.3.18 Inorganic ingredients 13 1.4 Results 14 1.4.1 Shortage of previous methods and new method development 14 1.4.2 Method validation through comparative analysis of organic/inorganic compounds 19 1.4.3 Method validation through cultivability of unculturable bacteria 21 1.4.4 Method validation through isolation rate of unculturable or new taxonomic bacteria 24 1.4.5 Method validation through taxonomic analysis 29 1.5 Discussion 33 Chapter II Taxonomical Characterization Of Novel Taxa 37 2.1 A Rapid And Simple Method For Identifying Bacterial Polar Lipid Components In Wet Biomass 37 2.1.1 Introduction 37 - ii - 2.1.2 Aim to study 38 2.1.3 Material and method 39 2.1.3.1 Organisms and growth conditions 39 2.1.3.2 Polar lipids extraction using the improved method 40 2.1.3.3 Polar lipids extraction using the the method of Minnikin et al (1984) 42 2.1.3.4 Polar lipids analysis 42 2.1.4 Results and Discussions 43 2.1.4.1 Polar lipid profiles of Microbacterium lacticum compared 43 2.1.4.2 Polar lipid profiles of Rhodococcus koreensis compared 44 2.1.4.3 Polar lipid profiles of Pseudomonas aeruginosa compared 48 2.1.4.4 Polar lipid profiles of Streptomyces longwoodensis compared 48 2.1.4.5 Polar lipid profiles of Novosphingobium capsulatum compared 48 2.2 Description of Flavobacterium fulvus sp nov., Flavobacterium pedocola sp nov., and Flavobacterium humicola sp nov., novel members belong to the family Flaviobacteriaceae, isolated from soil 51 2.2.1 Introduction 51 2.2.2 Materials and method 52 2.2.2.1 Sample and isolation of strains 52 2.2.2.2 Phenotypic characterisation 53 2.2.2.3 Genomic DNA isolate 54 2.2.2.4 Phylogenetic analyses 55 2.2.2.5 DNA–DNA hybridization (DDH) 56 2.2.2.6 G+C mol content 58 2.2.2.7 Determining fatty acids components 58 - iii - 2.2.2.8 Components of menaquinone and polar lipids 59 2.2.3 Results and discussion 61 2.2.3.1 Results of comparision between new isolates and their relative species 61 2.2.3.2 Description of novel isolates of genus Flavobacterium 72 Chapter III Isolation And Characterization Of Novel Antibiotic Candidates 78 3.1 General Information 78 3.1.1 Introduction 78 3.1.2 General background 79 3.1.2.1 Term of antibiotic 79 3.1.2.2 Antibiotic history 80 3.1.2.3 How antibiotics work to kill bacteria 82 3.1.2.4 Current problems with known drugs 86 3.1.2.5 Mechanisms of drug resistance 88 3.1.3 Goal of this study 91 3.2 Screening of Antibiotic-Producing Bacteria Isolated From Soils 92 3.2.1 Materials and methods 92 3.2.1.1 Soil sample 92 3.2.1.2 Isolation of microorganisms 92 3.2.1.3 For members of Actinobacteria 92 3.2.1.4 Modified culture method 93 3.2.2 Screening antibiotic-producing bacteria 93 - iv - 3.2.2.1 Microorganisms used to test 93 3.2.2.2 Assay of antimicrooganisms activities 94 3.2.2.3 Identification of 16S rRNA gene analysis 94 3.2.3 Results and discussion 94 3.2.3.1 Isolation of anti-microoganisms 94 3.2.3.2 Finding of the best strain to extract antibiotics 101 3.3 Characteristic Of Antibiotic Derived A Novel Species of Genus Sphingobium 104 3.3.1 Material and method 104 3.3.1.1 Fermentation and extraction of antibiotic 104 3.3.1.2 Purification of antibiotic extraction by silica gel column chromatography 105 3.3.1.3 Collection of individual compound by Prep-HPLC 105 3.3.1.4 Minimum inhibitory concentration (MIC) test 107 3.3.2 Results and discussion 108 3.3.2.1 Major descriptions of strain UCM-25 108 3.3.2.2 Extraction, purification, separation of antibiotics 110 3.3.2.3 Thin-Layer Chromatography (TLC) assay 110 3.3.2.4 Collecting individual compound 112 3.3.2.5 Minimal Inhibitory Concentration (M.I.C) test 116 3.4 An Antibiotic Producing Streptomyces Species Kills Drug Resistant Pathogens 118 3.4.1 Material and methods 118 3.4.2 Results and Discussion 119 -v- 3.4.2.1 Major information for strain T1317-0309 119 3.4.2.2 Screening of fractions collected by Prep-HPLC 126 3.4.2.3 Minimal Inhibitory Concentration (M.I.C) test 127 Chapter IV General Conclusion 131 4.1 Modification Of Soil Extraction Method 131 4.2 Taxonomical Characterization Of Novel Taxa 132 4.3 Isolation And Characterization Of Novel Antibiotic Candidates 133 Reference 135 Appendix 167 Abstract in Korean 202 - vi - List of Table Table Brief summary comparision of results by methods determined 15 Table List of soil extract components defined a new way and general approaches 17 Table List of bacterial isolates used for evaluating new soil extract (NSE) 21 Table Comparision of convenient methods for extracting bacterial polar lipids 49 Table Comparision of distinct phenotypic characteristics between four isolate strains and their type strains of the most related species of the genus Flavobacterium 63 Table Cellular fatty acid composition (%) of four isolate strains and the type strains of related species of the genus Flavobacterium 67 Table Antibiotic pathways to inhibit microoganisms 83 Table List of isolating bacterial soils that showed target microoganisms 95 Table Antibiotic extract activity of inhibition of target microoganisms using paper disc 102 Table 10 Activities of each fraction that are collected by Prep-HPLC, which inhibit target pathogens (Inhibition zone in mm) 113 Table 11 Minimal inhibitory concentration (MIC in μg ml-1) values of Fr-1 and Fr2 117 Table 12 Cultural characteristics of Streptomyces sp T1317-0309 on various media tested at 28oC for 21 days 120 Table 13 Result of identification of strain T1317-0309 based on 16S rRNA gene sequencing using the eztaxon-e 123 Table 14 Antibacterial activities of the Fr-5 collected from Streptomyces sp T13170309 129 - vii - Supplementary Table List of isolates obtained by modified method Strain/GenBank Accession Closely related to uncultured bacteria/Valid type species Similarity (%) Sample A: Sangnok-gu, Ansan-si, Suwon, Gyeonggi-do, South Korea MA1 (KY445600) Niabella soli DSM 19437T (AGSA01000025) DS-30T 97.5 MA3 Nocardioides hankookensis (EF555584) 98.7 MA83 Nocardioides hankookensis DS-30T (EF555584) 99.7 Uncultured bacterium clone 10-790 (KC554526) 98.1 Nocardioides panacisoli GSoil 346T (FJ666101) 96.7 MA7 (KY445601) RP-B26T MA13 (KY445603) Nocardioides ultimimeridianus (FM997998) 98.4 MA109 Nocardioides ultimimeridianus RP-B26T (FM997998) 99.8 KIS2-16T MA39 Nocardioides daecheongensis MA58 (KY445612) Nocardioides soli mbc-2T (JF937914) 616T (KJ135310) 99.7 98.0 MA67 Nocardioides panacihumi Gsoil MA4 Mycobacterium llatzerense MG13T (AJ746070) 99.9 MA5 Marmoricola solisilvae KIS18-7T (KM199638) 99.2 MA49 (KY445609) Amycolatopsis niigatensis LC11T (AB248537) 98.4 GXGD002T (AB271053) 99.0 MA9 Variovorax guangxiensis (JF495126) 98.7 MA48 Variovorax ginsengisoli Gsoil 3165T (AB245358) 98.7 MA15 MA46 (KY445608) Variovorax soli NBRC 106424T (BCUU01000176) Uncultured bacterium clone: GJ16S2_A07 (AB821140) 99.6 106424T 99.2 Variovorax soli NBRC (BCUU01000176) Uncultured bacterium clone SL24 (KF182268) MA12 (KY445602) 98.9 Paraburkholderia caryophylli ATCC 25418T (AB021423) 99.1 97.9 MA106 Paraburkholderia sartisoli RP007T (AF061872) 99.1 MA16 Rhodococcus maanshanensis M712T (AF416566) 100 MA20 Rhodococcus wratislaviensis NBRC 100605T (BAWF01000105) 99.9 MA116 Rhodococcus canchipurensis MBRL 353T (JN164649) 99.8 - 192 - MA126 Rhodococcus cercidiphylli YIM 65003T (EU325542) 98.7 MA17 (KY445604) Rhizobium tubonense CCBAU 85046T (EU256434) 97.5 MA19 Mycobacterium llatzerense MG13T (AJ746070) 99.5 MA51 Mycobacterium llatzerense MG13T (AJ746070) 99.7 MA22 Mycobacterium fluoranthenivorans FA-4T (AJ617741) 99.4 MA50 Mycobacterium septicum DSM 44393T (HG322957) 100 MA96 Microbacterium pumilum KV-488T (AB234027) 99.8 MA93 Microbacterium pumilum KV-488T (AB234027) 99.9 MA107 Microbacterium pumilum KV-488T (AB234027) 99.7 MA37 (KY445606) Microbacterium mangrovi MUSC 115T (JTDK01000032) 97.9 49404T MA42 Mycobacterium neworleansense ATCC MA45 Mycobacterium saopaulense EPM10906T (KM973037) 100 MA54 (KY445610) Microbacterium trichothecenolyticum DSM 8608T (JYJA01000006) 95.7 23848T (AY457068) MA62 Microbacterium azadirachtae DSM MA75 Mycobacterium rhodesiae DSM 44223T (AJ429047) 98.7 MA94 Microbacterium aoyamense KV-492T (AB234028) 99.3 MA123 Mycobacterium chlorophenolicum DSM 43826T (JYNL01000068) 99.9 MA23 Paenibacillus kribbensis AM49T (AF391123) 99.7 MA24 Leifsonia soli TG-S248T (EU912483) 99.3 MA102 Leifsonia shinshuensis JCM 10591T (DQ232614) 99.9 MA25 Rhodanobacter panaciterrae LnR5-47T (EU332829) 99.9 MA44 Rhodanobacter koreensis THG-DD7T (KF532124) 99.7 MA59 Rhodanobacter panaciterrae LnR5-47T (EU332829) 99.5 Uncultured Rhodanobacter sp clone DB-AN-204bp-TRF 99.3 MA91 (KY445618) (JYIT01000023) 99.9 99.5 (KM267483) Rhodanobacter panaciterrae LnR5-47T (EU332829) 99.3 MA30 Dyella japonica DSM 16301T (JPLA01000093) 100 MA60 Dyella marensis CS5-B2T (AM939778) 100 MA31 Terrabacter koreensis THG-e54T (KF314196) 99.6 - 193 - MA34 Methylobacterium soli YIM 48816T (EU860984) 99.6 MA38 (KY445607) Caballeronia zhejiangensis OP-1T (JFHD01000081) 98.6 Uncultured bacterium clone lp275 (KC331493) 99.4 Lysobacter panacisoli CJ29T (JQ806748) 98.4 Lysobacter ginsengisoli Gsoil 357T (AB245363) 99.7 Uncultured Lysobacter sp clone SBL06 (KM108693) 99.3 Lysobacter dokdonensis DS-58T (JRKJ01000003) 99.1 Uncultured gamma proteobacterium clone B6 (GQ249366) 99.1 Lysobacter lycopersici CC-Bw-6T (KC820654) 98.7 Uncultured bacterium clone D85 (KJ808189) 98.5 MA26 (KY445605) MA86 MA76 (KY445614) MA85 (KY445616) MA104 (KY445621) Lysobacter lycopersici CC-Bw-6T (KC820654) 98.2 MA47 Phycicoccus bigeumensis MSL03T (EF466128) 99.4 MA61 Phycicoccus bigeumensis MSL03T (EF466128) 99.9 MSL03T MA57 (KY445611) Phycicoccus bigeumensis (EF466128) 98.6 MA81 Phycicoccus bigeumensis MSL03T (EF466128) 98.7 MA55 Streptomyces nojiriensis LMG 20094T (AJ781355) 100 MA56 Mesorhizobium waimense ICMP 19557T (KC237387) 99.0 MA77 Mesorhizobium shonense AC39aT (GQ847890) 99.8 MA89 Mesorhizobium shangrilense CCBAU 65327T (EU074203) 100 MA63 Streptomyces tricolor NBRC 15461T (AB184687) 100 MA66 Streptomyces rishiriensis NBRC 13407T (AB184383) 99.7 MA71 Streptomyces brevispora BK160T (FR692104) 99.9 MA87 Streptomyces caeruleatus NRRL B-24802T (KQ948975) 99.5 MA99 Streptomyces amakusaensis NRRL B-3351T (AY999781) 99.9 MA64 Streptacidiphilus melanogenes NBRC 103184T (BBPP01000099) 99.7 MA68 (KY445613) Catenulispora graminis BR-34T (HM748599) 98.4 MA70 Cryptosporangium aurantiacum IFO 13967T (AB047490) 99.0 MA73 Sporosarcina aquimarina SW28T (AF202056) 99.4 - 194 - Uncultured bacterium clone ANTBIO-6 (KC138521) 99.9 Rhodopseudomonas faecalis gcT (AF123085) 98.7 MA82 Cohnella boryungensis BR-29T (HM061613) 99.9 MA90 (KY445617) Marmoricola solisilvae KIS18-7T (KM199638) 98.6 MA92 (KY445619) Nocardia niigatensis NBRC 100131T (BAGC01000015) 98.3 MA95 Bradyrhizobium jicamae PAC68T (LLXZ01000092) 99.9 MA108 Bradyrhizobium erythrophlei CCBAU 53325T (KF114645) 99.8 Uncultured soil bacterium clone A047 (JX489865) 98.9 Reyranella graminifolii Wo-34T (AB839882) 98.0 Collimonas pratensis Ter91T (CP013234) 99.0 CH15-11T 98.2 MA78 (KY445615) MA98 (KY445620) MA103 MA105 (KY445622) MA110 (KY445623) Sphingomonas daechungensis (JQ772481) Uncultured bacterium clone S23_830 (EF572731) 99.1 Ideonella dechloratans CCUG 30898T (X72724) 99.0 N-1T MA112 Cupriavidus necator (CP002878) 99.7 MA118 Cupriavidus necator N-1T (CP002878) 99.4 MA117 Bosea robiniae LMG26381T (FR774994) 99.5 MA120 (KY445624) Ralstonia pickettii ATCC 27511T (JOVL01000020) 97.5 MA124 Micromonospora chokoriensis 2-19/6T (AB241454) 100 Sample B: Buksu-dong, Paldal-gu, Suwon, Gyeonggi-do, South Korea MB2 (KY445625) Mycobacterium tokaiense ATCC 27282T (AF480590) 98.3 MB21 Microbacterium pumilum KV-488T (AB234027) 99.9 103464T MB30 Mycobacterium alvei CIP MB37 Mycobacterium neworleansense ATCC 49404T (AY457068) MG13T (AF023664) 99.7 99.9 MB55 Mycobacterium llatzerense (AJ746070) 99.9 MB82 Mycobacterium llatzerense MG13T (AJ746070) 98.9 MB77 (KY445637) MB81 MB73 (KY445635) MB94 (KY445641) Mycobacterium insubricum FI-06250T (EU605695) 98.3 Microbacterium mangrovi MUSC 115T (JTDK01000032) 99.7 Microbacterium mangrovi MUSC 115T (JTDK01000032) 98.0 Microbacterium mangrovi MUSC 115T (JTDK01000032) 97.9 - 195 - MB3 (KY445626) Gordonia kroppenstedtii NP8-5T (AM883151) NP8-5T MB11 Gordonia kroppenstedtii (AM883151) MB4 Flexivirga alba ST13T (AB539735) MB5 Nocardia acidivorans NBRC MB18 Nocardia altamirensis OFN S17T (EU006090) (BDAW01000136) MB87 (KY445638) Nocardia rayongensis (AB889540) MB6 Kitasatospora viridis 52108aT (AY613990) 0315T MB28 Kitasatospora nipponensis HKI MB60 Kitasatospora atroaurantiaca NRRL B-24282T (DQ026645) 15206T 99.3 100 108247T RY45-3T 98.1 (AY442263) 99.0 97.8 99.7 99.0 99.2 MB103 Kitasatospora kifunensis IFO MB8 Humibacillus xanthopallidus KV-663T (AB282888) 99.9 Uncultured bacterium clone RS-B18 (KC541072) 100 Pseudarthrobacter siccitolerans 4J27T (CAQI01000001) 99.3 MB10 (KY445627) MB12 (KY445628) Microvirga subterranea DSM 14364T (AB022874) 99.2 (FR733708) Uncultured actinomycete clone EHFS1_S10a (EU071503) MB14 (KY445629) MB74 (KY445636) MB109 (KY445646) MB33 Tetrasphaera japonica T1-X7T (HF570958) 99.9 98.2 99.2 98.1 Tetrasphaera duodecadis ATCC 13347T (AB072496) 97.7 Uncultured bacterium clone A3-392 (KC554848) 98.5 Tetrasphaera duodecadis ATCC 13347T (AB072496) 97.6 Mesorhizobium amorphae ACCC 19665T (AF041442) 99.8 Uncultured bacterium clone seqbac2-253 (KC191779) 99.4 Mesorhizobium tamadayense Ala-3T (AM491621) 98.6 Uncultured bacterium clone A1-63 (KC554727) 99.7 Marmoricola pocheonensis Gsoil 818T (GQ339906) 99.7 MB40 (KY445634) MB15 (KY445630) KIS18-7T MB16 Marmoricola solisilvae (KM199638) 99.2 MB34 Marmoricola solisilvae KIS18-7T (KM199638) 99.4 Uncultured bacterium clone D3-26 KC554916 98.1 Marmoricola bigeumensis MSL-05T (EF466120) 98.1 Uncultured Marmoricola sp clone BG2-174 (JX079253) 98.8 MB206 (KY445650) MB22 (KY445631) - 196 - Nocardioides iriomotensis IR27-S3T (AB544079) MB27 (KY445632) Nocardioides daecheongensis KIS2-16T (KJ135310) RP-B26T MB51 Nocardioides ultimimeridianus MB53 Nocardioides maradonensis RP-B30T (FM998000) MB107 Nocardioides paucivorans KIS31-44T Cr7-14T (FM997998) (KJ135311) MB202 Nocardioides alpinus (GU784866) MB24 Phycicoccus bigeumensis MSL03T (EF466128) 98.2 98.5 99.9 99.4 98.9 98.9 99.4 Phycicoccus bigeumensis MSL03T (EF466128) 99.3 MB59 Phycicoccus bigeumensis MSL03T (EF466128) 99.7 MB83 Phycicoccus bigeumensis MSL03T (EF466128) 99.9 MB58 Rhodanobacter panaciterrae LnR5-47T (EU332829) 98.8 MB76 Rhodanobacter panaciterrae LnR5-47T (EU332829) 99.9 MB52 Rhodanobacter koreensis THG-DD7T (KF532124) MB25 MB56 MB86 MB105 (KY445645) 99.8 Rhodanobacter panaciterrae LnR5-47T (EU332829) 100 Rhodanobacter panaciterrae LnR5-47T (EU332829) 99.9 Uncultured bacterium clone AMD-J13 (KC620912) 98.8 Rhodanobacter panaciterrae LnR5-47T (EU332829) 98.8 RP5557T MB71 Rhodanobacter lindaniclasticus MB102 (KY445644) Rhodanobacter soli DCY45T (FJ605268) 98.4 MB29 (KY445633) Luteimonas vadosa KMM9005T (AB704915) 96.7 Uncultured bacterium clone K2-15 (KC555002) 99.3 Streptomyces hundungensis MBRL 251T (JN560157) 99.2 Streptomyces anulatus NRRL B-2000T (DQ026637) 99.9 MB32 MB79 (AF039167) 13469T MB85 Streptomyces xanthocidicus NBRC MB91 Streptomyces hydrogenans NBRC 13475T (AB184868) 100 MB106 Streptomyces noboritoensis NBRC 13065T (AB184287) 100 MB110 MB95 (KY445642) Streptomyces hokutonensis R1-NS-10T (AB184427) 99.8 (BARG01000157) Streptomyces diastaticus subsp ardesiacus NRRL B-1773T (DQ026631) - 197 - 99.7 100 98.3 MB36 MB38 Rhodopseudomonas rhenobacensis Klemme RbT (AB087719) Humibacillus xanthopallidus KV-663T 15500T 98.7 (AB282888) 99.0 MB39 Sphingomonas mali NBRC (BCYX01000023) 100 MB201 (KY445648) Sphingomonas soli NBRC 100801T (BCTZ01000019) 98.1 MB57 Caballeronia cordobensis LMG 27620T M712T (FCNY01000054) MB70 Rhodococcus maanshanensis (AF416566) MB204 Rhodococcus fascians LMG 3623T (JMEN01000010) Diaminobutyricibacter tongyongensis KIS66-7T MB75 Streptacidiphilus melanogenes NBRC 103184T MB80 Ralstonia mannitolilytica LMG 6866T (AJ270258) MB72 MB205 (KY445649) MB207 (KY445651) MB84 MB104 MB89 (KY445639) MB92 MB93 (KY445640) Ralstonia pickettii ATCC 27511T (JX876865) (BBPP01000099) 99.6 100 98.9 99.9 99.9 99.0 (JOVL01000020) 97.5 Uncultured bacterium clone 300A-F12 (AY662035) 99.7 Ralstonia syzygii subsp celebesensis UQRS 627T (KC757073) 97.7 Bacillus gibsonii DSM 8722T Bacillus butanolivorans DSM (X76446) 99.8 18926T 99.9 (LGYA01000001) Uncultured Ideonella sp clone CD35 (KJ191921) 99.4 Ideonella dechloratans CCUG 30898T (X72724) 98.6 Bradyrhizobium erythrophlei CCBAU 53325T (KF114645) 99.8 Uncultured Lysobacter sp clone SBL06 (KM108693) 99.4 Lysobacter dokdonensis DS-58T (JRKJ01000003) 99.0 PY2T MB96 Terrabacter carboxydivorans MB97 Rhodoferax saidenbachensis ED16T (AWQR01000064) 98.8 Uncultured bacterium clone ANTBIO-6 (KC138521) 99.9 Rhodopseudomonas faecalis gcT (AF123085) 98.7 Arthrobacter bambusae GM18T (KF150696) 99.2 Uncultured bacterium clone: GJ16S2_A07 (AB821140) 99.7 Variovorax soli NBRC 106424T (BCUU01000176) 99.3 Uncultured bacterium clone D43 (JX271938) 99.2 MB99 (KY445643) MB108 MB111 (KY445647) MB208 (KY445652) - 198 - (FJ717334) 98.9 Sphingobium phenoxybenzoativorans SC_3T (KP257600) MB209 (KY445653) Cellulomonas terrae DB5T (AY884570) 99.1 98.6 Sample S: Itaewon-ro, Yongsan-gu, Seoul, South Korea MS3 Bacillus thuringiensis ATCC 10792T (ACNF01000156) 18422T MS4 Bacillus luciferensis LMG MS27 Bacillus toyonensis BCT-7112T (CP006863) 100 MS72 Bacillus drentensis LMG 21831T (AJ542506) 100 MS5 MS28 MS19 MS6 (KY445654) (AJ419629) 100 100 Marmoricola solisilvae KIS18-7T (KM199638) 99.9 Marmoricola solisilvae KIS18-7T (KM199638) 99.7 097T Marmoricola ginsengisoli Gsoil (GQ339892) Uncultured bacterium, clone FB02B03 (FM872945) Terrabacter terrae PPLBT 99.7 99.8 (AY944176) 98.8 20308T MS7 Terrabacter tumescens DSM (X83812) 99.0 MS25 Terrabacter carboxydivorans PY2T (FJ717334) 98.8 Uncultured bacterium clone RS-B50 (KC541104) 98.3 MS11 (KY445656) MS13 (KY445657) MS23 MS67 MS8 (KY445655) MS15 (KY445659) MS41 MS45 (KY445667) Pseudarthrobacter oxydans KCTC (X83408) Uncultured bacterium clone 11-145 (KC554588) 98.1 99.9 Pseudarthrobacter siccitolerans 4J27T (CAQI01000001) 99.2 Pseudarthrobacter siccitolerans 4J27T (CAQI01000001) 99.0 Pseudarthrobacter siccitolerans 4J27T (CAQI01000001) 99.4 Uncultured bacterium clone 4-504 (KC554243) 98.8 Nocardioides dubius KSL-104T (AY928902) 97.3 Nocardioides panacihumi Gsoil 616T (AB271053) 98.5 Nocardioides halotolerans DSM Nocardioides daecheongensis 19273T KIS2-16T MS65 (KY445672) Nocardioides panacihumi Gsoil MS66 (KY445673) T MS79 (KY445678) 3383T 616T (KE383922) 98.8 (KJ135310) 98.0 (AB271053) Nocardioides aquiterrae GW-9 (AF529063) Nocardioides caricicola YC6903T MSL03T 98.5 98.3 (FJ750845) 98.6 (EF466128) 99.9 MS10 Phycicoccus bigeumensis MS18 Arthrobacter pascens DSM 20545T (X80740) 99.7 MS21 (KY445660) Arthrobacter pascens DSM 20545T (X80740) 97.7 MS22 MS61 MS24 Arthrobacter humicola KV-653T (AB279890) 100 Arthrobacter humicola KV-653T (AB279890) 99.8 12137T 99.4 Arthrobacter globiformis NBRC - 199 - (BAEG01000072) MS80 Arthrobacter globiformis NBRC 12137T (BAEG01000072) 99.7 MS95 Arthrobacter globiformis NBRC 12137T (BAEG01000072) 100 MS26 (KY445661) MS64 (KY445671) MS68 MS70 (KY445674) Arthrobacter cupressi D48T Arthrobacter enclensis (HQ657321) NIO-1008T 98.6 (JF421614) 98.3 Arthrobacter ramosus CCM 1646T (AM039435) 99.4 Arthrobacter ramosus CCM 1646T (AM039435) 98.6 Arthrobacter bambusae GM18T (KF150696) 99.3 MS96 Arthrobacter bambusae GM18T (KF150696) 99.4 MS98 Arthrobacter bambusae GM18T (KF150696) 99.9 MS73 Arthrobacter oryzae NRRL B-24478T (AB279889) 99.9 MS71 MS54 (KY445669) MS14 (KY445658) MS16 MS20 Arthrobacter livingstonensis LI2T (GQ406811) 98.3 Uncultured Vogesella sp clone A8 (JF833455) 99.3 Crenobacter luteus YIM 78141T Ipa-2T Pseudomonas mohnii Caballeronia humi LMG (KF771276) 94.6 (AM293567) 22934T 99.0 (FCNW01000121) 27620T MS69 Caballeronia cordobensis LMG MS29 Agromyces iriomotensis IY07-20T (AB546308) 99.5 MS30 Mycobacterium rhodesiae DSM 44223T (AJ429047) 99.2 MS31 MS99 MS100 MS33 MS44 (KY445666) MS37 (KY445664) MS42 MS90 MS62 (KY445670) MS34 (KY445662) Mycobacterium aichiense ATCC (FCNY01000054) 99.9 27280T 99.6 (X55598) 99.0 Mycobacterium phocaicum CIP 108542T (AY859682) 99.0 Mycobacterium phocaicum CIP 108542T (AY859682) 99.1 Lysobacter lycopersici CC-Bw-6T (KC820654) Uncultured gamma proteobacterium clone B6 (GQ249366) Lysobacter lycopersici CC-Bw-6T (KC820654) 99.9 99.5 99.2 Uncultured Lysobacter sp clone T92c06 (HM447669) 99.7 Lysobacter dokdonensis DS-58T (JRKJ01000003) 96.0 Lysobacter ginsengisoli Gsoil Lysobacter niabensis 357T GH34-4T (AB245363) (DQ462461) 100 Uncultured Lysobacter sp clone T312E2 (HM438543) Lysobacter panacisoli CJ29T Bradyrhizobium jicamae (JQ806748) PAC68T 99.7 99.1 98.8 (LLXZ01000092) 98.3 53363T MS92 Bradyrhizobium guangxiense CCBAU (KC508877) 99.9 MS38 Bradyrhizobium erythrophlei CCBAU 53325T (KF114645) 99.1 MS35 (KY445663) Povalibacter uvarum Zumi 37T (AB548216) 97.5 - 200 - MS36 MS43 (KY445665) MS52 (KY445668) MS56 MS49 Streptosporangium jomthongense 30EHST (JQ922513) 99.5 Uncultured Rhodanobacter sp clone T2-34 (KM200382) 99.3 Rhodanobacter umsongensis LC11T Amycolatopsis niigatensis Amycolatopsis echigonensis Gordonia kroppenstedtii GR24-2T (AB248537) LC2T NP8-5T (FJ821731) 99.0 98.4 (AB248535) 99.2 (AM883151) 99.3 13-12-50T 100 MS48 Actinomadura maheshkhaliensis MS51 Catenulispora graminis BR-34T MS81 Catenulispora yoronensis TT N02-20T (AB327250) 99.7 MS55 Nocardia salmonicida NBRC 13393T (BDBR01000081) 99.6 MS76 MS53 MS77 MS82 (AB331731) (HM748599) Nocardia niigatensis NBRC 100131T Streptomyces amakusaensis NRRL B-3351T Streptomyces graminisoli JR-19T 98.8 (BAGC01000015) (AY999781) (HQ267975) 40451T Streptomyces olivochromogenes DSM 99.8 99.9 99.9 (KQ948511) 99.9 Streptomyces vulcanius NEAU-C3T MS97 Streptomyces exfoliatus NRRL B-2924T MS57 Streptacidiphilus rugosus AM-16T (JQMJ01000004) 99.9 MS58 Dyella marensis CS5-B2T (AM939778) 100 MS83 (KY445679) MS63 MS74 (KY445675) MS93 (KY445683) MS75 (KY445676) Variovorax soli NBRC 106424T (KF956803) (JNZP01000081) (BCUU01000176) Paenibacillus vulneris CCUG 53270T Paenibacillus hemerocallicola DLE-12T (HE649498) (JQ723718) Uncultured Microlunatus sp clone: DCP26-4 (LC093422) Microlunatus phosphovorus NM-1T (AP012204) 85046T 99.9 99.2 96.6 97.0 98.9 98.8 MS78 (KY445677) Rhizobium tubonense CCBAU MS84 Burkholderia contaminans LMG 23361T (LASD01000006) 99.7 Uncultured Bacteroidetes bacterium clone YH102 (JQ861407) 99.8 MS85 (KY445680) MS86 (KY445681) MS91 (KY445682) MS94 Flavitalea populi HY-50RT (EU256434) 98.6 (HM130561) Uncultured Ideonella sp clone CD35 (KJ191921) Aquincola tertiaricarbonis L10T (DQ656489) Uncultured bacterium clone 2-7C (EU289504) Methylobacterium cerastii Cupriavidus necator N-1T C44T (FR733885) (CP002878) - 201 - 97.4 98.1 99.7 98.4 97.7 97.5 99.1 국문요지 박사학위논문 난배양성 토양세균의 분리 및 항생제 후보물질의 확보 경기대학교 대학원 생명과학과 누엔만투안 난배양성 토양세균의 수많은 대사산물의 잠재력은 매우 크다 그 동안 수 많은 방법들이 이러한 난배양성 토양세균을 찾기 위해 개발돼왔지만 이러한 방법들은 풀어야 할 장애물이 있는데 이 장애물은 난배양성 세균의 수 많은 종들이 자랄 수 있는 고형배지의 부재에 있다 본 연구는 수 많은 토양세균의 본고장과 같은 토양에서 유래된 새로운 토양추출물(NSE)을 통해 더 많은 호기 성 난배양성 토양세균을 찾기 위해 고안되었다 본 연구에서 개발한 NSE는 기존의 증류수를 이용한 토양추출물과 달리 증류수와 메탄올 혼합물을 이용했 다는 점이 다르고 더 많은 난배양성 토양세균을 분리할 수 있었다 더욱더 이 방법을 이용하면 기존의 다른 배양방법에 비해 쉽고 시간을 절약할 수 있으며 집적배양이 필요 없고 전통배지와 혼합해서 사용 할 수 있다 화학분석결과 NSE는 유기화학영양세균, 무기화학영양세균, 종속영양세균 을 포함한 토양미생물에게 필요한 주요 구성성분들을 포함하고 있었으며, 몇 가지 활성 성분을 추가하여 새로운 배지인 ISEM (Intensive Soil Extraction Medium for unculturable soil bacteria)을 만들었다 ISEM을 통해 7개의 문(phylum) - 202 - 에 속하는 균주들을 성공적으로 배양하였는데, 총 258개 분리균주 중에 약 49% 가 기존의 난배양성세균에 속하였고 약 55%가 종(species), 속(genus) 또는 과 (family)수준의 새로운 분류후보군으로 판별되었다 그러므로 본 연구는 다양한 토양환경에 감추어진 수 많은 세균의 성공적인 배양을 통한 새로운 원리와 지 식을 발굴하는 초석이 될 것으로 기대된다 비록 극성지질분석에 동결건조세포를 사용하여 성공적으로 수행해왔지만 습윤세포(wet cell)를 사용한 몇몇 방법들이 개발되어왔지만 습윤세포를 이용할 경우 축출된 인지질의 비극성 때문에 적용에 많은 한계점을 보여주었다 본 연구에서는 그람양성과 그람음성 균을 포함한 5개의 종 즉 Microbacterium lacticum, Rhodococcus koreensis, Pseudomonas aeruginosa, Streptomyces longwoodensis 그리고 Novosphingobium capsulatum의 습윤세포를 이용하여 지질분석을 수행하 여 몇몇 소소한 성분을 제외하고 기존의 동결건조법과 유사한 결과를 도출해 냈다 더욱이 이 개선된 방법은 지질추출과정을 단순화시겼다 그람음성, 내성포자비생성, 비이동성인 균주들을 대전시 유성구의 토양으 로부터 분리하였다 16S rRNA 유전자의 염기서열을 통해 이 균주들은 Flavobacterium속에 속하는 것으로 판명되었고, 이 중 균주 UCM-R15T와 UCMR21는 F enshiense DK69T (97.38-97.46%), F saliperosum S13T (96.28-96.35%), F suncheonense GH29-5T (96.28-96.35%), F limnosediminis JC2902T (96.14-96.22%), F cauense R2A-7T (95.17-95.67%), F columnare IFO 15943T (94-88-95.25%)와 UCMR36T는 F suncheonense GH29-5T (97.53%), F enshiense DK69T (96.57%), F limnosediminis JC2902T (96.43%), F saliperosum S13T (96.29%), F cauense R2A-7T (95.74%)와 UCM-46T는 F suncheonense GH29-5T (98.27%), F cauense R2A-7T (96.06%), F enshiense DK69T (95.5%), F saliperosum S13T (95.44%), and F limnosediminis JC2902T (95.44%)와 가장 가까운 유연관계를 보여주었다 4균주 모 - 203 - 두 질산염(nitrate)과 요소(urea)를 분해하지 못했고 주요 menaquinone는 MK-6였 고, 주요 지방산은 C15:0 iso, C17:0 iso 3-OH, C15:1 iso G, summed feature (C17:1 iso ω7c and/or C16:0 10-methyl, Phosphatidylethanolamine는 C15:0 세 iso 3-OH, 균주에서 C15:0 주요 anteiso, 극성지질로 C16:0 iso였다 분석되었고, phosphatidylserine는 균주 UCM-R15T와 UCM-R36T에서는 발견되었으나 UCM46T에서는 발견되지 않았다 한편Phosphatidylmonomethylethanolamine는 UCMR15T에서만 발견되었고 더욱이 미동정된 L2 또는 L3는 160-180°C에서 15분 후에 나타났으나 나머지는 빠르게 나타났다 DNA G+C Mol%는 UCM-R15T, UCM-R21, UCM-R36T, UCM-46T 별로 각각 35.3, 36.2, 39.0, 38.4였다 DNA-DNA hybridization 결과는 분리된 균주와 비교균주사이에 70%이하를 보여주었고 생 리적 및 생화학적 특성을 접목하여 3개의 균주가 Flavobacterium속에 속하고 각각의 이름을 Flavobacterium fulvus, Flavobacterium pedocola, Flavobacterium pedocola로 제안한다 각각의 Type균주명은 UCM-R15T (=KACC 18666T =NBRC 111764T), UCM-R36T (=KACC 18668T =NBRC 111765T), UCM-46T (=KACC 18575T =NBRC 111657T)로 한다 2013년에 한국전역의 산림에서 100개 이상의 토양시료를 채취해서 3,000 개 이상의 분리균주를 얻었고 이중에 43개가 미국부저병(AFB)의 원인균인 Paenibacillus larvae 및 일반 병원균인 Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans and Aspergillus niger에 대한 향균력을 보여주었 다 16S rRNA 유전자 서열분석을 통해 이들은 12개의 속에 속한다는 것을 알게 되었고 대부분 방선균들 이었다 속들은 많은 것부터 순서대로 Streptomyces (28), Kitasospora (4), Bacillus (2), Streptacidiphilus, Amycolatopsis, Paenibacillus, Promicromonospora, Rhodococcus, Pseudomonas, Burkholderia, Undibacterium, Actinomadura였다 이들 중에 Streptomyces속의 종들이 65.12%로 가장 많았고 Burkholderia와 Undibacterium 종들은 그람양성균에 대한 항균력을 가지는 것으 - 204 - 로 본 연구에서 처음으로 보고하였다 이들 중에 그 동안 항균작용이 보고되 지 않았던 Sphingobium속에 속하는 균주 UCM-25에 집중하여 신종으로 분류하 여 Sphingobium aromaticivorans로 명명하고 배양액 추출물의 prep-HPLC의 분취 액 Fr-1과 Fr-2의 항균력을 조사하였다 Bacillus subtilis KEMB 51201-001 (16; 64 μg ml-1, MIC respectively), Staphylococcus aureus KEMB 4659 (16; 64 μg ml-1), Pseudomonas aeruginosa KACC 10185 (16; 128 μg ml-1), Escherichia coli KEMB 212234 (32; 128 μg ml-1), Paenibacillus larvae KACC 14031 (16; 32 μg ml-1), Streptococcus agalactiae CCARM 4504 (64; 128 μg ml-1), Streptococcus pyogenes CCARM 4520 (32; 256 μg ml-1), Enterococcus faecalis CCARM 5168 (128; 128 μg ml-1), Enterococcus faecalis CCARM 5171 (64; 128 μg ml-1) 한국의 한 토양시료에서 분리한 균주 T1317-0109는 그람양성균에는 강한 활성을 나타내나 그람음성, 효모 및 곰팡이류에는 활성이 없었고 16S rRNA 유전자 염기서열분석을 통해 Streptomyces속에 속하는 균주로 판명되었다 PrepHPLC의 분취액 Fr-5가 물에 잘 녹지는 않지만 다른 용매 즉, 메탄올, 에탄올, 에틸아세톤에는 잘 녹았고 이는 TLC분석과 prep-HPLC peak 순서를 통해서도 분취액에 녹아있는 분자가 비극성임에 가까움을 알 수 있었다 MIC test 결과 항생제 내성 균주를 포함한 다양한 그람음성 병원성 균주에 효과가 있는 것으로 나타났다 Bacillus subtilis KEMB 51201-001 (0.5 μg per ml), Bacillus anthracis KEMB 211-146 (0.5), Staphylococcus aureus KEMB 4659 (1), Paenibacillus larvae KACC 14031 (0.125), Streptococcus agalactiae CCARM 4504 (0.5), Streptococcus pyogenes CCARM 4520 (0.5), Enterococcus faecalis CCARM 5168 (0.5), Enterococcus faecalis CCARM 5171 (0.5), Enterococci faecalis CCARM 5025 (0.5), Enterococci faecium CCARM 5024 (0.5), Staphylococcus epidermidis KACC 13234 (1), methicillinresistant Staphylococcus aureus CCARM 3095 (0.5), methicillin-resistant Staphylococcus aureus CCARM 3192 (4), methicillin-resistant Staphylococcus aureus CCARM 3155 (1.25), methicillin-resistant Staphylococcus aureus CCARM 3710 (1.25), methicillinresistant Staphylococcus aureus CCARM 3A041 (1.25), Propionibacterium acnes subsp - 205 - acnes KCTC 3314 (0.25)에 효과가 있었다 이러한 결과는 본 연구에서 순수분리한 항생제 후보물질이 일부 항생제 내성 균주에서 MIC 값이 상용화된 항생제보다 더 낮게 나옴으로 더 효과적인 항생제 개발에 사용될 가능성이 있음을 나타낸다 - 206 - .. .Isolation Of Unculturable Soil Bacteria And Finding Of Their Antibiotic Candidates by Nguyen Manh Tuan A dissertation submitted to the faculty of the Graduate School in partial fulfillment of. .. thank you all of Professors at the Department of Life Science, Kyonggi University: Prof Byung-Sun Yoon, Prof Sang Seob Lee, Prof Dong-Soo Kong, Prof Byung-Sun Yoo, Prof Ok-Min Lee, and Prof Seong-Ho... Information of soil property used for the method 1.3.2 Preparing new soil extraction (NSE) in litre medium 1.3.3 Medium for isolation of soil bacteria 1.3.4 Soil sampling site and preparation