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1 Korean Journal of Microbiology (2016) Vol. 52, No. 3, pp pissn DOI eissn Copyright c 2016, The Microbiological Society of Korea Note 제주도인근해양퇴적물내의미생물군집구조분석 고현우 라니선다스 황한빛 박수제 * 제주대학교생물학과 Microbial community structure analysis from Jeju marine sediment Hyeon Woo Koh, Sundas Rani, Han-Bit Hwang, and Soo-Je Park* Department of Biology, Jeju National University, Jeju 63243, Republic of Korea (Received July 7, 2016; Revised August 2, 2016; Accepted August 8, 2016) ABSTRACT: In this study, the structure and diversity of bacterial community were investigated in the surface and subsurface marine sediments using a NGS method (i.e. illumina sequencing technology). The bacterial community in the surface was distinct from that in the subsurface of marine sediment; with the exception of the phylum Proteobacteria, the relative abundance of Bacteroides phylum were higher in the surface than subsurface, whereas the sequences affiliated to the phyla Chloroflexi and Acidobacteria were relatively more copious in the subsurface than surface sediment. Moreover, interestingly, we observed that the phyla Nitrospinae and Nitrospirae contribute to nitrogen cycle in the marine sediment. This study may present the possibility for the presence of novel microorganisms as unexplored sources and provide basic information on the microbial community structure. Key words: bacteria, community, marine sediment, next-generation sequencing 미생물은지구상에서인간의삶을포함한모든부분에서필수적인요소이며, 생지화학적순환을담당하고있다. 하지만대부분의미생물들은실험실에서배양이어려운한계때문에배양에의존하지않는분자생물학적분석이주를이루고있다 (Amann et al., 1995). 차세대염기서열분석 (next-generation sequencing, NGS) 기술의발달이래, 16S ribosomal RNA (rrna) 유전자를활용한미생물의다양성및군집구조분석은기존의 clone library, T-RFLP, DGGE 등의기법으로부터빠르게대체되고있다 (Hamady et al., 2008; Caporaso et al., 2011). 해양환경은지구표면적의약 70% 를차지하고있으며, 열수구 (hydrothermal vent) 를포함하는심해 (deep-sea) 와같은환경을포함하고있다. 해양미생물들은다양한환경에적응하기위하여독특한물질대사와생리적능력을지니고있는것으로알려져있다. 또한, 이들미생물의종풍부도와다양성은매우높은것으로확인되고있으며 (Walsh et al., 2016), 그들은전지구적인생지화학순환을담당하고있는것으로알려져있다 (Choi et al., 2016). 해양미생물의생지화학적순환에기 *For correspondence. sjpark@jejunu.ac.kr; Tel.: ; Fax: 여하는정확한종다양성및그들이지닌대사과정에대한연구는활발히이루어지고있으나, 아직까지도미비한실정이다. 특히최근에발효된생물다양성협약 ( 나고야의정서 ) 에따라, 국내서식하는미생물들의종다양성파악및발굴이가속화되고있다. 이에따라, 본연구에서는국내의대표적인섬인제주도해양퇴적물내의미생물다양성및군집구조를 NGS 기법을통하여분석하였다. 본실험을위하여, 해양퇴적물은제주모슬포인근 (33 13 N, E) 에서시료를채취하였다. 퇴적물시료는상층부 (0 10 cm) 와하층부 (10 30 cm) 로구분하여, 멸균된 50 ml conical tube에담아, 실험전까지 -80 C에보관하였다. 각시료는 Power Soil TM DNA kit (Mo Bio Laboratories) 를이용하여제조사의방법에따라 genomic DNA (gdna) 를추출하였으며, 추출된 gdna는전기영동장치에 0.8% (w/v) 아가로즈젤을이용하여 gdna의크기와분해여부를확인하고, DS-11 plus spectrophotometer (Denovix, Inc.) 를이용하여농도를확인하였다. NGS 분석을위하여, 미생물의 16S ribosomal RNA (16S rrna) 유전자의 V4 영역을 PCR 증폭및정제하였다 (Kim et al., 2015). 정제된 PCR 산물의 NGS 수행은 Macrogen

2 376 Koh et al. 에의뢰하였으며, MiSeq (illumina, Inc.) 장비를사용하였다. NGS 결과로얻어진염기서열들은 Mothur (version ) 프로그램을이용하여분석하였으며, 얻어진서열의길이가짧거나긴것, 그리고키메릭서열들은제거하였다. 또한, Ribosomal Database Project (RDP) database를통하여미생물이외의염기서열 ( 미토콘드리아와엽록체 ) 들도제거하여, 분석의신뢰도를향상하였다. 종 (species) 수준의분류와, operational taxonomic unit (OTU) 를포함하는미생물의다양성은 mother 프로그램에서제공하는알고리즘을이용하여 97% 유사도를기준으로분석하였다 (Kim et al., 2015; Koh et al., 2015). 전처리를거친후, 상 하층부의해양퇴적물로부터최종적으로각각, 약 13,000와 19,000여개의염기서열을확보하여분석하였으며, Good s coverage는평균 75% 로확인되었다. 다양성지수를비교한결과, 하층부의해양퇴적물내에미생물다양성, 우점도및풍부도가상층보다높음을확인할수있었다. 하지만, 다양도분석결과 (-index) 둘간의차이 ( 상층, 6.98; 하층, 7.47) 가크지않아서보다많은샘플수를이용하여분석의정확도를높이는것이필요할것으로판단된다. 미분류균 (unclassified group) 으로분류된염기서열을포함하여, 409개의종들이 29개의문으로분류됨을확인하였다. 미분류군은다양한환경에서분자생물학적분석 ( 비배양적분석 ) 을통하여얻어진서열만보고된미생물분류군을칭한다 (McDonald et al., 2012). 흥미롭게도, 상층퇴적물에서미분류균으로분류된염기서열들은약 9.6% 임에비하여하층의경우 31% 로이들에대한정보량이높아졌음을확인할수있었다. 이러한사실로미루어볼때, 혐기적환경으로파악되는하층부의해양퇴적물에지금까지분류되지않은미생물분류군이존재하고있을것으로판단된다. 문 (phylum) 수준에서, 상층퇴적물의경우 Proteobacteria (69.8%) 와 Bacteroides (11.8%) 가우점하는것에비하여, 하층부의경우, Proteobacteria (45.0%), Chloroflexi (8.1%), 그리고 Acidobacteria (6.4%) 로확인되었다. 해양퇴적물내의질소순환에기여하는것으로알려져있는 Nitrospinae 와 Nitrospirae 문도상 하층퇴적물모두서식하고있는것으로확인되었다. 이러한점을바탕으로대부분모래등으로구성되어미생물의다양성이낮을것으로예측되는제주해양퇴적물과는달리모슬포인근해양퇴적물은생지화학적물질순환에기여할것으로판단되는기초적정보를제공하나, 배양혹은환경유전체학적분석을통하여이를보다정밀하게분석할필요가있다. 상층퇴적물에서 Proteobacteria 문을제외하고, 우점하는문의경우대부분방향족화학물, 탄화수소류를포함하는난분해성물질들을분해하는것으로알려져있는 Bacteroides 문이였다 (Roling et al., 2002; Vinas et al., 2005). 게다가, 통성혐기적호흡을통하여생태계내에서황순환혹은난분해성물질을분해하는특징을지닌것으로확인되는 Chloroflexi 문의경우 (Yamada and Sekiguchi, 2009), 하층퇴적물에서 8.1% 로우점하는것으로관찰되었다. 이러한사실로미루어, 상 하층의해양퇴적물내에서난분해성유기물분해능이높다고판단된다. Proteobacteria 문중에서, 상 하층퇴적물에서모두 Alpha-, Delta- 와 Gammaproteobacteria강이우점하였으나, 상층부에서는 Gammaproteobacteria강이하층부에서는 Deltaproteobacteria 강이우점하는것으로확인되었다. 이는퇴적물내의이용가능한용존산소와관련이있을것으로판단된다. 해양환경내의 Gammaproteobacteria강의경우주로호기적호흡을통하여에너지를획득하는반면에, Deltaproteobacteria강은다양한유 무기물을이용하여혐기적호흡을통하여에너지를획득하는미생물이 (e.g. sulfate-reducing Deltaproteobacteria) 다수발견되고있는것과연관된다고할수있다 (Miyatake et al., 2009; da Silva et al., 2013). 또한, 본연구에서는 Aminicenantes, Hydrogenedentes문및후보문 (candidate division) 인 WPS-2와 ZB3을포함하여, pyrosequencing을통한미생물군집구조를분석한기존연구에서 (Choi et al., 2016) 발견되지않은희박생물권 (rare biosphere) (Lynch and Neufeld, 2015) 의미생물들도확인되었다. 강 (class) 수준에서도 pyrosequencing에비하여 MiSeq 플랫폼을이용한본연구를통해보다다양한미생물분류군들이확인되었다. 미분류군속에분류된염기서열을제외하고, 속 (genus) 수준에서는 Desulfobacteraceae 과의미분류속에속하는염기서열이 11.7% 로하층퇴적물에서는우점하였으나, 상층퇴적물의경우 Gammaproteobacteria 강의미분류속에속하는것이 23.9% 로우점하는것으로확인되었다. 그리고, 예상과다르게 Mollicutes 강을포함하는 Tenericutes 문이상층퇴적물에서매우적은양으로검출되었다 (Brown et al., 2007). Mycoplasma 가속하는이문은세포벽이없으며, 대부분숙주세포에기생하는병원성미생물로알려져있으며, 일반적인해양퇴적물에서발견된다. 이들은해양환경내에서산호등과같은해양무척추동물들과연관이있을것으로판단되므로 (Neulinger et al., 2008, 2009; Kellogg et al., 2009), 해양퇴적물뿐만아니라해양무척추동물들내의미생물군집구조분석에대한추가적인연구도필요하다. 게다가, 최근심해의메탄공 (deep-sea methane seep) 퇴적물로부터 Tenericutes 문과관련된분류군인 NB1-n 과 RF3의유전체들로부터분석되었다. 이유전체는수소화효소 (hydrogenase) 와 cytochrome bd oxidase와 complex I으 미생물학회지제 52 권제 3 호

3 해양퇴적물의미생물군집 377 로구성된단순한전자전달계가발견되었으며, Candidatus Izimaplamsa sp. HR1과 HR2로각각명명되었다 (Skennerton et al., 2016). 이러한사실에미루어볼때, Tenericutes 문에대한자연환경에서의생태학적기능에대한연구는현재까지매우미비한실정이다. 본연구에서는 pyrosequencing을통한기존연구에서는발견되지않았던보다다양한미생물분류군들이발견되었다. 이는 NGS 분석을위한플랫폼의차이보다는 16S rrna 유전자분석영역의차이때문인것으로사료된다. 비록 16S rrna 유전자의 V3/V4와 V4/V5 영역이 454와 illumina의플 랫폼을사용한 in silico 분석에서다른영역에비하여높은분류정확도를보이긴하지만, 실제실험에서는 V3/V4 영역의편향된증폭현상 (amplification bias) 이관찰된다고보고된바있다 (Claesson et al., 2010). 그럼에도불구하고, V4를포함하는영역은 NGS 분석에있어매우적합하다고판단된다. 또한, Kuczynski 등 (2012) 은환경시료와인간미생물군 (human microbiome) 시료에대해서각각 V4 영역과 V1-V2 영역분석을제안하기도하였다. 본연구에서도 Good s coverage를제외하고, 기존의 pyrosequencing에의하여분석된미생물의다양성지수를비교한결과 (Table 1), 큰차이를볼수없었으나, Fig. 1. The abundance of the phyla in Bacteria in the marine sediments. The bacterial 16S rrna gene sequences were assigned to each phylum using the mothur package and a reference database of 16S rrna genes obtained from Ribosomal Database Project. Table 1. Estimation of sequence diversity and operational taxonomic unit (OTU) of the surface and subsurface marine sediments. Diversity was estimated using OTUs and was defined as groups with 3% sequence dissimilarity. Diversity indices were calculated by mothur package. Sample Analyzed reads Observed OTUs lci a hci a lci hci lci hci Good s coverage Surface 12,638 4, , , , Subsurface 18,860 6, , , , a lci and hci are rarefied 95% low and high confidence intervals (provide by the mothur application), respectively. Korean Journal of Microbiology, Vol. 52, No. 3

4 378 Koh et al. 희박미생물 (rare microorganism) 과같은미생물분류군의존재확인에있어의미있는차이를관찰할수있었다. 해양퇴적물로부터확보한대부분의염기서열들은미분류군들과상동성을보였으며이것은아직까지해양퇴적물에서식하는많은미생물들이배양되지않았다는것을의미한다. 따라서이러한미생물들의배양을통하여, 새로운분류군으로분류될수있으며그들의생태학적기능을유추할수있을것이다. 세균과고세균들은 rrna 분석을통하여 60여개의문들이보고되었으나, 이들중배양된균주들이속하는문은절반정도에지나지않는다 (Hugenholtz and Kyrpides, 2009; Rinke et al., 2013). 따라서해양퇴적물내에특성이밝혀지지않은미생물들이우점한다는점에서새로운토착미생물자원으로이용될수있는가능성이존재한다. 본연구를통하여, 해양퇴적물내의미생물군집구조와그다양성을비교 분석하였으며, 이결과는해양미생물자원발굴에대한기초자료를제공할수있을것이라사료된다. 적요 본연구에서는차세대염기서열분석기법을활용하여제주도인근해양퇴적물의상층부와하층부내의미생물군집구조와다양성을조사하였다. 상층부와하층부의미생물군집구조는상이하였으며, Proteobacteria 문을제외하고, 상층부에서는 Bacteroides 문이, 하층부에서는 Chloroflexi와 Acidobacteria 문이각각우점하는것으로확인되었다. 또한, 흥미롭게도질소순환에관여하는것으로알려진 Nitrospinae와 Nitrospirae 문도서식하고있음이관찰되었다. 본연구를통하여, 아직발굴되지않은새로운미생물의자원으로서의가능성과해양퇴적물내의기본적인정보를제공할수있을것으로기대한다. 감사의말 이논문은 2016년도제주대학교교원성과지원사업에의하여연구되었습니다. References Amann, R.I., Ludwig, W., and Schleifer, K.H Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, Brown, D.R., Whitcomb, R.F., and Bradbury, J.M Revised minimal standards for description of new species of the class Mollicutes (division Tenericutes). Int. J. Syst. Evol. Microbiol. 57, Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Lozupone, C.A., Turnbaugh, P.J., Fierer, N., and Knight, R Global patterns of 16S rrna diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. USA 108 Suppl 1, Choi, H., Koh, H.W., Kim, H., Chae, J.C., and Park, S.J Microbial community composition in the marine sediments of Jeju island: next-generation sequencing surveys. J. Microbiol. Biotechnol. 26, Claesson, M.J., Wang, Q., O'Sullivan, O., Greene-Diniz, R., Cole, J.R., Ross, R.P., and O'Toole, P.W Comparison of two nextgeneration sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rrna gene regions. Nucleic Acids Res. 38, e200. da Silva, M.A.C., Cavalett, A., Spinner, A., Rosa, D.C., Jasper, R.B., Quecine, M.C., Bonatelli, M.L., Pizzirani-Kleiner, A., Corção, G., and Lima, A.O.d.S Phylogenetic identification of marine bacteria isolated from deep-sea sediments of the eastern South Atlantic Ocean. SpringerPlus 2, Hamady, M., Walker, J.J., Harris, J.K., Gold, N.J., and Knight, R Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat. Methods 5, Hugenholtz, P. and Kyrpides, N.C A changing of the guard. Environ. Microbiol. 11, Kellogg, C.A., Lisle, J.T., and Galkiewicz, J.P Culture-independent characterization of bacterial communities associated with the cold-water coral Lophelia pertusa in the northeastern Gulf of Mexico. Appl. Environ. Microbiol. 75, Kim, Y.S., Kim, J., and Park, S.J High-throughput 16S rrna gene sequencing reveals alterations of mouse intestinal microbiota after radiotherapy. Anaerobe 33, 1 7. Koh, H.W., Kim, M.S., Lee, J.S., Kim, H., and Park, S.J Changes in the swine gut microbiota in response to porcine epidemic diarrhea infection. Microbes Environ. 30, Kuczynski, J., Lauber, C.L., Walters, W.A., Parfrey, L.W., Clemente, J.C., Gevers, D., and Knight, R Experimental and analytical tools for studying the human microbiome. Nat. Rev. Genet. 13, Lynch, M.D. and Neufeld, J.D Ecology and exploration of the rare biosphere. Nat. Rev. Microbiol. 13, McDonald, D., Price, M.N., Goodrich, J., Nawrocki, E.P., DeSantis, T.Z., Probst, A., Andersen, G.L., Knight, R., and Hugenholtz, P An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 6, Miyatake, T., MacGregor, B.J., and Boschker, H.T Linking microbial community function to phylogeny of sulfate-reducing Deltaproteobacteria in marine sediments by combining stable isotope probing with magnetic-bead capture hybridization of 16S rrna. Appl. Environ. Microbiol. 75, 미생물학회지제 52 권제 3 호

5 해양퇴적물의미생물군집 379 Neulinger, S.C., Gartner, A., Jarnegren, J., Ludvigsen, M., Lochte, K., and Dullo, W.C Tissue-associated Candidatus Mycoplasma corallicola and filamentous bacteria on the cold-water coral Lophelia pertusa (Scleractinia). Appl. Environ. Microbiol. 75, Neulinger, S.C., Jarnegren, J., Ludvigsen, M., Lochte, K., and Dullo, W.C Phenotype-specific bacterial communities in the coldwater coral Lophelia pertusa (Scleractinia) and their implications for the coral s nutrition, health, and distribution. Appl. Environ. Microbiol. 74, Rinke, C., Schwientek, P., Sczyrba, A., Ivanova, N.N., Anderson, I.J., Cheng, J.F., Darling, A., Malfatti, S., Swan, B.K., Gies, E.A., et al Insights into the phylogeny and coding potential of microbial dark matter. Nature 499, Roling, W.F., Milner, M.G., Jones, D.M., Lee, K., Daniel, F., Swannell, R.J., and Head, I.M Robust hydrocarbon degradation and dynamics of bacterial communities during nutrient-enhanced oil spill bioremediation. Appl. Environ. Microbiol. 68, Skennerton, C.T., Haroon, M.F., Briegel, A., Shi, J., Jensen, G.J., Tyson, G.W., and Orphan, V.J Phylogenomic analysis of Candidatus Izimaplasma species: free-living representatives from a Tenericutes clade found in methane seeps. ISME J. DOI Vinas, M., Sabate, J., Espuny, M.J., and Solanas, A.M Bacterial community dynamics and polycyclic aromatic hydrocarbon degradation during bioremediation of heavily creosote-contaminated soil. Appl. Environ. Microbiol. 71, Walsh, E.A., Kirkpatrick, J.B., Rutherford, S.D., Smith, D.C., Sogin, M., and D'Hondt, S Bacterial diversity and community composition from seasurface to subseafloor. ISME J. 10, Yamada, T. and Sekiguchi, Y Cultivation of uncultured Chloroflexi subphyla: significance and ecophysiology of formerly uncultured Chloroflexi subphylum i with natural and biotechnological relevance. Microbes Environ. 24, Korean Journal of Microbiology, Vol. 52, No. 3

example code are examined in this stage The low pressure pressurizer reactor trip module of the Plant Protection System was programmed as subject for

example code are examined in this stage The low pressure pressurizer reactor trip module of the Plant Protection System was programmed as subject for 2003 Development of the Software Generation Method using Model Driven Software Engineering Tool,,,,, Hoon-Seon Chang, Jae-Cheon Jung, Jae-Hack Kim Hee-Hwan Han, Do-Yeon Kim, Young-Woo Chang Wang Sik, Moon