Korean Journal of Microbiology (2016) Vol. 52, No. 1, pp. 65-73 pissn 0440-2413 DOI http://dx.doi.org/10.7845/kjm.2016.6015 eissn 2383-9902 Copyright c 2016, The Microbiological Society of Korea 보문 한강물로부터분리된방사선내성세균들의계통학적다양성및 UV 내성분석 이재진 1 주은선 1 이도희 1 정희영 2 김명겸 1 * 1 서울여자대학교자연과학대학생명환경공학과, 2 경북대학교농업생명과학대학응용생명과학부 Phylogenetic diversity and UV resistance analysis of radiation-resistant bacteria isolated from the water in Han River Jae-Jin Lee 1, Eun Sun Joo 1, Do Hee Lee 1, Hee-Young Jung 2, and Myung Kyum Kim 1 * 1 Department of Bio & Environmental Technology, College of Natural Science, Seoul Women s University, Seoul 01797, Republic of Korea 2 College of Agricultural and Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea (Received March 4, 2016; Revised March 21, 2016; Accepted March 22, 2016) ABSTRACT: The aim of this study was to investigate the UV-resistance of radiation-resistant bacteria isolated from the water of Han River, South Korea. The water sample was irradiated with 3 kgy gamma radiation prior to isolation. Radiation-resistant bacterial strains were isolated by standard serial dilution method on R2A and 1/10 diluted R2A agar. The resulting purely isolated 60 cultures of bacteria were analysed for UV resistance and used in further studies. Based on the comparative analyses of 16S rrna gene sequences, the bacterial isolates were divided into 3 phyla (4 genera): the phylum Deinococcus-Thermus (the genus Deinococcus) was 61.7%, Bacteroidetes (Hymenobacter and Spirosoma) was 23.4%, and Firmicutes (Exiguobacterium) was 15%. The results suggested that twenty-nine isolates are candidates new species belonging to Deinococcus, Hymenobacter, and Spirosoma, or other new genera. Nine bacterial strains were selected among the novel candidates and the UV-resistance analysis was conducted. All the candidate bacterial strains showed high UV resistance, similar to that of D. radiodurans R1. Key words: 16S rdna, Han River, phylogenetic tree, radiation-resistant bacteria, UV resistance analysis, water Deinococcus radiodurans 균주 (Brooks and Murray, 1981) 가처음으로발견되면서방사선내성미생물에대한관심이생기게되었다. 이들이가지고있는 DNA 복구효소의활성원리를응용한노화방지연구, 암세포의방사선내성감소를위한내성유전자연구, 방사선내성생물체개발을통한오염된환경의생물학적정화등과같은가능성들이밝혀지게되면서 (Cox and Battista, 2005; Servinsky and Julin, 2007; Kwon and Seo, 2010), 이러한방사선내성연구를위한방사선내성미생물자원확보가더많이요구되고있는추세이다. Deinococcus와 Rubrobater 가대표적으로방사선내성이강한속 (genus) 으로알려져있으며, 이보다방사선내성이약한속으로는 Acinetobacter, Chroococcidiopsis, Hymenobacter, *For correspondence. E-mail: biotech@swu.ac.kr; Tel.: +82-2-970-5667; Fax: +82-2-970-5974 Kineococcus, Kocuria, Methylobacterium, Pyrococcus, Thermococcus 이있는것으로밝혀졌다 (Rainey et al., 2005; Yu et al., 2015). 최근에는 Spirosoma 속이 Deinococcus 만큼의강한내성을보이며 (Lee et al., 2014; Lee et al., 2015), 계속새로운다양한방사선내성미생물이밝혀지고있는중이다. 그리고이들은방사선뿐만이아니라 UV와건조한환경에도잘견디는것으로밝혀졌다 (Mattimore and Battista, 1996; de Groot et al., 2005). 방사선내성미생물은남극, 북극, 사막, 방사선에오염된토양과같은극한환경에서얻는경우가많으며 (Hirsch et al., 1998; 2004; Yuan et al., 2009; Wang et al., 2010), 그외에도공기, 강물, 토양, 실험실, 나무, 바다고기, 목재섭식흰개미등다양한환경에서분리되었다 (Lai et al., 2006; Zhang et al., 2007; Im et al., 2008; Shashidhar and Bandekar, 2009; Yoo et al., 2010; Cha et al., 2014).
66 Lee et al. 이렇게방사선내성균주가분리되었던다양한환경들중에서물에서분리된방사선내성미생물의보고가상당수이루어짐에따라 (Im et al., 2008; Kämpfer et al., 2008; Asker et al., 2009; 2011), 우리나라서울한강물에서방사선내성을가지는것으로추측되는미생물을분리및탐색하고, 16S rdna 유전자염기서열을비교하여한강에서의방사선내성미생물의계통수를분석하고자하였다. 또한 2016년 2월현재까지우리나라한강물에서분리되어신종으로보고된균주들은 Brevibacillus fluminis CJ71 T, Chryseobacterium rigui CJ16 T, Paenibacillus aestuarii CJ25 T 등총 3건 (Suresh et al., 2004; Bae et al., 2010; Choi et al., 2010; Park et al., 2013) 으로더많은신종미생물보고를위하여신종연구가가능한것으로추정되는신종예상균주를파악하여 UV 내성추가실험을통해 UV 내성여부를분석하고방사선내성연구에필요로하는유용미생물자원을확보하고자하였다. 재료및방법 한강물채집, 농축, 방사선조사및세균의분리 2016년 1월에여의도공원근처의한강상층수를멸균된병에 1 L 담아서채집한후, Hydrophilic membrane filter (Mixed Cellulose ester 02047A, 0.20 μm, Hyundai Micro.) 로여과하여 10 ml 증류수에여과된필터지를넣어균질화하였다. 균질화된시료에 3 kgy의감마선 (gamma-ray) 을조사하고연속희석법으로희석한후, 현탁액을 R2A agar (Difco) 및 1/10로희석된 R2A agar (Difco) 배지에각각도말하여 25 C에 7일간배양하였다. 3 kgy 방사선에견디고자라난군락 (colony) 을군락의모양, 크기, 색깔등에따라선별하여처음에도말했던평판배지에계대배양하여순수분리하였다. 분리된미생물은 20% (v/v) glycerol에현탁하여초저온냉동고 (-80 C) 에동결보관하였다. Genomic DNA 추출, 16S rdna 유전자증폭및염기서열분석 Genomic DNA는 InstaGene Matrix (Bio-Rad) 시약을사용하여분리하였다. 먼저평판배지에균주를접종하여배양후 colony를취하여마이크로튜브안 1 ml 증류수에용해시킨후원심분리 (10,000 12,000 rpm, 1분 ) 해서상등액을제거하고, InstaGene Matrix를 50 μl 첨가후 56 C에서 15분간반응시켰다. 튜브를 10초동안빠른속도로 vortexing하고 10분동안 100 C에끓인후, 다시튜브를 10초동안빠른속도로 vortexing하고원심분리 (10,000 12,000 rpm, 3분 ) 하여 16S rrna 유전자분석을위한 genomic DNA를추출하였다. 16S rrna 유전자를증폭하기위해 27F (5 -AGAGTTTGATCM TGGCTCAG-3 ) primer와 1492R (5 -TACGGYTACCTTG TTACGACTT-3 ) primer를사용하였고, MJ Research 사의 PTC-225 Peltier Thermal Cycler를사용하여 PCR 반응은초기변성 (95 C, 5분 ), 변성 (95 C, 1분 ), 냉각 (55 C, 1분 ), 신장 (72 C, 1분 ) 을 35회반복한후마지막신장 (72 C, 5분 ) 을실시하였다. Applied Biosystems 사의 ABI 3730XL DNA analyzer 를이용하여유전자염기서열결과를얻었다. 이러한유전자염기서열분석은 마크로젠 (Macrogen) 을통해이루어졌다. 계통학적분석분석된염기서열은 EzBioCloud의 database (http://www. ezbiocloud.net/eztaxon/) 를이용해서 GenBank로부터유사성이높은염기서열을비교하여가장가까운종으로나타나는서열을확인하였다. 본연구에선택된균주들의 16S rrna 염기서열 alignment는 BioEdit (Hall, 1999) 으로수집, Clustal_X (Thompson et al., 1997) 를이용하여정렬한후에 MEGA 5.0 software (Molecular Evolutionary Gnetics Analysis; Tamura et al., 2011) 을이용하여 neighbor-joining 방법 (Saitou and Nei, 1987) 에의해계통수를추론하였다. Ultra-Violet 내성실험배양된균주를 0.85% NaCl에연속희석후, 희석된균체를 R2A agar에 10 μl씩접종하였다. Negative control로는 Escherichia coli K-12 (=KCTC 1116) (Kämpfer et al., 2008), Positive control로는 Deinococcus radiodurans R1 T (=DSM 20539 T ) (Brooks and Murray, 1981; White et al., 1999) 을사용하였다. 접종된 R2A agar plate의뚜껑을열고 UVC ultraviolet crosslinker (UVP, CX-2000) 를사용하여 254 nm의파장으로 UV를조사하였다. 선량률 (dose rate) 는 50 J/m 2 /sec이며, 노출시간을조정함에따라전체 UV 조사량이달라진다. UV 조사후, 25 C에서 3일간배양한다음 colony를계수하였다. 결과및고찰 배지에따른세균수및분리균주의균주수방사선조사된한강수샘플로부터평판배양법으로세균수를측정한결과, 평균적으로 R2A 평판배지에서분리한방 미생물학회지제 52 권제 1 호
방사선내성미생물 67 사선내성세균은 6.7 10 5 CFU/ml였고, 1/10 R2A 평판배지에서는 1.4 10 5 CFU/ml의세균수가관찰되었다. R2A 보다 1/10 R2A 배지에서세균수가낮게계수되었다. 이렇게해서방사선이조사된한강물로부터순수분리한균주는총 60개였고, 각배지별로얻어진균주수를살펴보면, R2A 평판배지에서 42개, 1/10 R2A 평판배지에서 18개균주를얻었다 (Table 1A). 염기서열분석및계통학적분석방사선이조사된한강물에서순수분리된 60개균주의 16S rrna 유전자염기서열을가지고 EzbioCloud를이용하여계통학적다양성을분석해본결과, Deinococcus-Thermus 문 (Deinococcus 속 ), Firmicutes 문 (Exiguobacterium 속 ), Bacteroidetes 문 (Hymenobacter, Spirosoma 속 ) 의 3개의문, 4개의속, 13개의종으로동정되었다. 이중 Deinococcus-Thermus 문이총 37개 (61.7%) 균주로가장많이나타났고, 그다음은 Bacteroidetes (14개; 23.4%), Firmicutes (9개; 15%) 문의순으로분포하였다 (Table 1B). R2A 평판배지에서생장한 42개균주중 Deinococcus spp. 은 21개 (50%) 로절반을차지하였고, Exiguobacterium spp. 와 Hymenobacter spp. 은각 9개씩 ( 각 21.4%), Spirosoma spp. 은 3 개 (7.1%) 였다. 1/10 R2A 평판배지에서는 Deinococcus spp. 가 16개 (88.9%) 로대부분을차지하였다 (Table 1A). 각배지에서분리된 Deinococcus spp. 의수가큰차이가없는것으로보아 Table 1. Diversity of cultivable bacteria from the gamma-radiated water sample of Han River in Seoul (total isolates = 60) (A) Genera of bacterial isolates on (1) R2A and (2) 1/10 R2A media from the water of Han River. (B) Diversity of bacterial isolates of the gamma-radiated water of Han River. (A) (1) Medium : R2A (2) Medium : 1/10 R2A Genus Number Ratio (%) Number Ratio (%) Deinococcus sp. 21 50.0 16 88.9 Exiguobacterium sp. 9 21.4 0 0 Hymenobacter sp. 9 21.4 1 5.6 Spirosoma sp. 3 7.1 1 5.6 Total 42 100.0 18 100.0 (B) Phylum Order Genus Species Number Ratio (%) Deinococcus-Thermus Deinococcales Deinococcus aquaticus 29 48.3 aquatilis 3 5.0 daejeonensis 3 5.0 grandis 2 3.3 Total 37 61.7 Firmicutes Bacillales Exiguobacterium acetylicum 2 3.3 sibiricum 7 11.7 Total 9 15.0 Bacteroidetes Cytophagales Hymenobacter algoricola 4 6.7 elongatus 3 5.0 gelipupuascens 1 1.7 rigui 1 1.7 terrae 1 1.7 Total 10 16.7 Spirosoma endophyticum 3 5.0 rigui 1 1.7 Total 4 6.7 The Sum Total 60 100.0 Korean Journal of Microbiology, Vol. 52, No. 1
68 Lee et al. Table 2. Identification of radiation-resistant bacteria isolated from the waters of Han River in Seoul Medium R2A 1/10 R2A Phylum Deinococcus- Thermus Closest relative based on 16S rrna gene sequnces Isolated name GenBank Accession No. Simiarity (%) Deinococcus aquaticus PB314 T Deinococcus aquaticus 16F3N KU758886 99.52 Deinococcus aquaticus PB314 T Deinococcus sp. 16F7B* processing 98.15 Deinococcus aquaticus PB314 T Deinococcus sp. 16F1E* processing 97.79 Deinococcus aquaticus PB314 T Deinococcus sp. 16F3H* processing 97.65 Deinococcus aquaticus PB314 T Deinococcus sp. 16F3J* processing 97.33 Deinococcus daejeonensis MJ27 T Deinococcus sp. 16F1L KU758885 98.40 Firmicutes Exiguobacterium acetylicum DSM 20416 T Exiguobacterium acetylicum 16F3E KU758892 99.60 Exiguobacterium acetylicum DSM 20416 T Exiguobacterium acetylicum 16F3I KU758893 99.60 Exiguobacterium sibiricum 255-15 T Exiguobacterium sibiricum 16F5B KU758894 99.60 Exiguobacterium sibiricum 255-15 T Exiguobacterium sibiricum 16F5C KU758895 99.60 Exiguobacterium sibiricum 255-15 T Exiguobacterium sibiricum 16F1F KU758891 99.54 Bacteoidetes Hymenobacter algoricola VUG-A23a T Hymenobacter sp. 16F7C* KU758881 97.88 Deinococcus- Thermus Hymenobacter elongatus VUG-A112 T Hymenobacter elongatus 16F7A KU758896 99.05 Hymenobacter elongatus VUG-A112 T Hymenobacter sp. 16F3P* KU758879 98.46 Hymenobacter gelipurpurascens Txg1 T Hymenobacter gelipurpurascens 16F7D KU758897 99.38 Hymenobacter rigui WPCB131 T Hymenobacter sp. 16F7G* KU758880 97.42 Hymenobacter terrae DG7A T Hymenobacter sp. 16F3Y* KU758882 97.74 Spirosoma endophyticum EX36 T Spirosoma sp. 16F3U KU758883 92.83 Spirosoma rigui WPCB118 T Spirosoma rigui 16F3V KU758898 99.93 Deinococcus aquatilis DSM 23025 T Deinococcus aquatilis 16F6B KU758889 99.16 Deinococcus aquatilis DSM 23025 T Deinococcus aquatilis 16F6I KU758890 98.76 Deinococcus aquatilis DSM 23025 T Deinococcus aquatilis 16F4D KU758888 98.63 Deinococcus grandis DSM 3963 T Deinococcus grandis 16F4B KU758887 98.67 Bacteoidetes Spirosoma endophyticum EX36 T Spirosoma sp. 16F6E* KU758884 92.90 * UV-radiation resistance test were done (Fig. 2) 저영양배지인 1/10 R2A에서방사선내성이높다고알려진 Deinococcus 균주를분리하기가더욱용이함을알수있었고, Deinococcus 이외의다른속에속한종들을얻고자할때에는 R2A 배지가 1/10 R2A 배지보다더용이하였다. 16S rrna 유전자염기서열분석에따르면 Deinococcus- Thermus 문인 Deinococcus 속에속하는균주는기존의염기서열과 97.2 99.5% 의상동성을나타내었고, D. aquaticus PB314 T, D. aquatilis DSM 23025 T, D. daejeonensis MJ27 T, D. grandis DSM 3963 T 와각각 97.2 99.5, 98.6 99.2, 98.4 99.2, 98.7-99.0% 의유사성을보여주었다. Firmicutes 문인 Exiguobacterium 속에속해있는균주는대부분기존종의표준균주염기서열과 99.1 99.7% 의높은상동성을보여주었으며, 16F3E, 16F3I는 E. acetylicum DSM 20416 T 과 99.6%, 16F1F, 16F5B, 16F5C는 E. sibiricum 255-15 T 와 99.5 99.6% 의높은유사도를나타내었다 (Table 2). 그리고 Bacteroidetes 문에속하는균 주는기존종의표준균주염기서열과 92.8 99.9% 의상동성을보여주었는데, Bacteroidetes 문의 Spirosoma 속에속하는 16F3U, 16F6E는 S. endophyticum EX36 T 와 92.8 92.9% 의낮은염기서열유사도를보여주었고, 163V는 Spirosoma rigui WPCB118 T 와 99.9% 의높은유사도를나타내었다 (Table 2). Bacteroidetes 문의 Hymenobacter 속분리균주들은 H. algoricola VUG-A23a T, H. elongatus VUG-A112 T, H. gelipurpurascens Txg1 T, H. rigui WPCB131 T, H. terrae DG7A T 와각각 97.2 98.1%, 98.5 99.4%, 99.4%, 97.4%, 97.7% 의상동성이확인되었다. 이와같이 16S rrna 유전자염기서열비교를통해분리된 60개균주중 26개균주는기존종의표준균주와 97 98.5% 상동성을보여추후신종실험에의해신종가능성을보이며, 3개균주는 92.8 93.0% 의상동성을보여신종실험을통해새로운속과종으로서의가능성이있다고판단됨으로추가적인신종연구를필요로한다 (Woo et al., 2008). 이러한연구 미생물학회지제 52 권제 1 호
방사선내성미생물 69 를통해지금까지한강물에서밝혀지지않았던다수의신속및신종미생물연구를진행할수있을것으로기대된다. 이렇게분리된균주 (60개) 중에서신종가능성이있는균주 (29개중에서 ) 11개 (Fig. 1-붉은색 ) 와기존에밝혀진종과유사도가높은균주 (31개중에서 ) 13개 (Fig. 1-녹색 ) 를선정하여, 이들총 24개균주를 NCBI의 GenBank에등록하고 (Table 2), 계통학적분석을통하여계통수를작성한결과 Fig. 1과같다. 본연구에서분리된균주들과상동성이높은표준균주 Deinococcus aquaticus PB314 T, Deinococcus aquatilis DSM 23025 T 등은강물이나산업지역물 (industry water) 에서분리되었고 gamma선과 UV에내성을가지고있는것으로나타났다 (Im et al., 2008; Kämpfer et al., 2008). 또한 Exiguobacterium sibiricum 255-15 T 은시베리안의영구동토 (permafrost) 에서분리되었고 (Rodrigues et al., 2006), Hymenobacter elongatus VUG-A112 T 는빅토리아빙하에서분리되었으며 (Klassen and Fought, 2011), Spirosoma rigui WPCB118 T 는우포늪의물에서분리되었다 (Baik et al., 2007). 이러한결과로보아한강에서분리된균주들과유전적상동성이높은표준균 Fig. 1. Phylogenetic Neighbor-joining tree based on comparison of the 16S rrna gene sequences of bacteria isolated from the gamma-radiated water sample of Han River and representatives of three phylum (A, Deinococcus-Thermus; B, Firmicutes; C, Bacteoidetes). Boostrap values (>50%) based on 1,000 replications are shown. Bar 0.02 nucleotide substitutions per nucleotide position. Red color, novel species; Green color, already known species. Korean Journal of Microbiology, Vol. 52, No. 1
70 Lee et al. Fig. 1. Continued. 주대부분은물이나극한지의빙하와관련된시료로부터얻어진것을알수있었다. Ultra-Violet 내성분석신종가능성을가지는분리균주들중에서 9개의신종균주들을선정하여실제 UV 세기에따른생장여부를확인하기위해서 UV 내성실험을진행하였다. Deinococcus 속에속하는신종후보균주인 16F1E, 16F3H, 16F3J 등 3균주는 D 10 값 ( 미생물의개체수가 10분의 1로줄어들때방사선조사량 ) 이 600 J/m 2 이하이며, 이균주들은방사선내성이높다고알려진 positive control로사용한 Deinococcus radiodurans R1 T 균주만큼높은 UV 내성결과를보였고, 16F7B 균주는 D 10 값이 300 J/m 2 이하로나타났다. Hymenobactr 신종의경우, 3개의 16F3P, 16F7C, 16F7G 균주는 D 10 값이 600 J/m 2 이하이며, 이균주들 도 D. radiodurans R1 T 균주와비슷하게높은 UV 내성결과를보였고, 그중 16F7G 균주는전체적인그래프가 D. radiodurans R1 T 균주보다더높은 UV 내성결과를보였다. 16F3Y 균주는 D 10 값이 500 J/m 2 이하로나타났다. Spirosoma 신종균주 16F6E 는 D 10 값이 900 J/m 2 이하이며 D. radiodurans R1 T 균주보다전체적으로더높은 UV 내성결과를보였다 (Fig. 2). 결론적으로, 9개의신종균주모두 UV 내성을가지는것으로나타났고, 대부분은방사선내성이높다고알려진 D. radiodurans R1 T 균주만큼높은 UV 내성결과를보였고, Hymenobacter sp. 16F7G와 Spirosoma sp. 16F6E 균주는 D. radiodurans R1 T 보다더높은내성을보이기도하였다. 본연구를통하여분리된균주들중에서 Deinococcus 균주가절반이상을차지하는결과와현재까지밝혀진방사선내성표준균주들이물에서분리된경우가많은것으로볼때 미생물학회지제 52 권제 1 호
방사선내성미생물 71 신종균주는아니었지만분리균주인 Firmicutes 문에속한 Exiguobacterium 속의균주는아직까지방사선내성연구보고가되어있지않아서이에대한연구가더욱필요하며, 특히우리나라의한강물에서식하는세균의군집구조에관한보고는거의없기때문에이에관한연구가필요한실정이다. 적요 이논문은서울한강물에서분리한방사선내성세균군집과분리된신종세균의 UV 내성특성에관한내용이다. 세균은 R2A agar와 1/10 R2A agar를사용하여 3 kgy가조사된한강물에서분리되었다. 그결과방사선에내성을가지는것으로추측되는균주를 60주분리하였고, 본연구분석자료로사용하였다. 16S rrna 유전자염기서열분석을통해분리균주 60 개의계통수를파악한결과, 3개의문 (4개의속 ) 이확인되었고, Deinococcus-Thermus (Deinococcus) 가 61.7%, Firmicutes (Exiguobacterium) 는 15%, Bacteroidetes (Hymenobacter, Spirosoma) 는 23.4% 의비중을나타냈다. 분리균주중 29개균주가 Deinococcus, Hymenobacter, Spirosoma에속하는신종또는다른신속으로분류될가능성을보였으며, 앞으로추가적인신종실험이진행될예정이다. 그리고신종예상균주를 9개선정하여 UV 내성실험을진행한결과, 9개균주모두 D. radiodurans R1 T 균주만큼높은 UV 내성을가지는것으로나타났다. 또한분리된 Firmicutes (Exiguobacterium) 균주는아직까지방사선내성연구보고가되어있지않아서추가적인방사선내성연구가필요하다. 감사의말 이논문은 2016학년도서울여자대학교교내학술연구비의지원을받았음. Fig. 2. Survival rates of (A) Deinococcus sp. 4 strains (16F1E, 16F3H, 16F3J, 16F7B), (B) Hymenobacter sp. 4 strains (16F3P, 16F3Y, 16F7C, 16F7B), (C) Spirosoma sp. strain 16F6E after irradiation with UVC radiation at 254 nm. Deinococcus radiodurans R1 (positive control), and Escherichia coli strain K12 (negative control) served as positive and negative control, respectively. The y-axis shows survival on a logarithmic scale. (Gabani and Singh, 2013), 한강물에서도방사선내성균주가많이존재할가능성이있다고보여진다. 그리고본연구에서 References Asker, D., Awad, T.S., Beppu, T., and Ueda, K. 2009. Deinococcus aquiradiocola sp. nov., isolated from a radioactive site in Japan. Int. J. Syst. Evol. Microbiol. 59, 144 149. Asker, D., Awad, T.S., McLandsborough, L., Beppu, T., and Ueda, K. 2011. Deinococcus depolymerans sp. nov., a gamma and UV-radiation-resistant bacterium, isolated from a naturally radioactive site. Int. J. Syst. Evol. Microbiol. 61, 1448 1453. Korean Journal of Microbiology, Vol. 52, No. 1
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