Korean Journal of Microbiology (2017) Vol. 53, No. 4, pp pissn DOI eissn Copyright

Korean Journal of Microbiology (2017) Vol. 53, No. 4, pp. 251-256 pissn 0440-2413 DOI https://doi.org/10.7845/kjm.2017.7066 eissn 2383-9902 Copyright c 2017, The Microbiological Society of Korea Article 식물생장을촉진할수있는구리내성세균의분리 김민주 송홍규 * 강원대학교생명과학과 Isolation of copper-resistant bacteria with plant growth promoting capability Min-Ju Kim and Hong-Gyu Song* Department of Biological Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea (Received September 4, 2017; Revised October 24, 2017; Accepted October 26, 2017) Some rhizobacteria were isolated, that have copper resistance and can confer copper resistance to plants allowing growth under copper stress. Isolated strains Pseudomonas veronii MS1 and P. migulae MS2 produced 0.13 and 0.26 mmol/ml of siderophore, that is a metal-chelating agent, and also showed 64.6 and 77.9% of biosorption ability for Cu in 20 mg/l Cu solution, respectively. Copper can catalyze a formation of harmful free radicals, which may cause oxidative stress in organisms. Removal activity of 1,1-diphenyl-2-picryl hydrazyl radical and antioxidant capacity of strains MS1 and MS2 increased up to 82.6 and 78.1%, respectively compared to those of control at 24 h of incubation. They exhibited 7.10 and 6.42 µmol α-ketobutyrate mg/h of 1-aminocyclopropane-1-carboxylic acid deaminase activity, respectively, which reduced levels of stress hormone, ethylene in plants, and also produced indole- 3-acetic acid and salicyclic acid that can help plant growth under abiotic stress. All these results indicated that these copper-resistant rhizobacteria could confer copper resistance and growth promotion to plants. Keywords: ACC deaminase, antioxidant capacity, Cu-resistant bacteria, plant growth promotion, siderophore *For correspondence. E-mail: hgsong@kangwon.ac.kr; Tel.: +82-33-250-8545; Fax: +82-33-259-5665 구리는생물체의생존을위해요구되는필수미량원소이지만구리가함유된물질의과다한사용이나폐기물투기등에의해구리농도가높아지면모든생물체에독성을나타내게된다 (Cervantes and Gutierrez-Corona, 1994). 중금속이유입되면토양에흡착되며분해되지않기때문에축적되어식물에스트레스로작용하여생장이억제되고농업에피해를유발하기때문에식물생장을유지하고촉진할수있는방법이필요하다. 또한금속오염토양자체의정화를위한식물환경복원방법의일환으로과축적식물 (hyperaccumulator) 을이용하는식물추출법 (phytoextraction) 의핵심도식물의금속에대한내성과빠른생장이다 (Abou-Shanab et al., 2006; Pandey et al., 2013). 식물이금속, 건조, 염분등의비생물적스트레스를받게되면이에대한반응으로 S-adenosylmethionine에서 1-aminocyclopropane-1-carboxylic acid (ACC) 를거쳐식물호르몬인에틸렌이합성되며이의농도가높아지면생장을억제하고노화, 성숙등을유도하기때문에스트레스에틸렌이라부른다 (Morgan and Drew, 1997). 이런스트레스반응시식물생장을지속시키는방법중하나로식물생장촉진근권세균 [plant growth promotiong rhizobacteria (PGPR)] 을이용할수있다. 이들은세포표면에금속을흡착하고, 금속과결합하는 siderophore를생성하여금속의흡수를막거나, 다양한방법으로금속으로인한산화스트레스를감소시킨다 (Cervantes and Gutierrez-Corona, 1994). 한편 ACC deaminase를분비하여에틸렌의농도를낮추어

252 Kim and Song 식물에금속스트레스에대한내성을부여하고 (Glick et al., 1998), 대표적인식물생장촉진호르몬인 indole-3-acetic acid (IAA) 등의분비, 가용한질소나인의공급등의방법으로식물의생장을촉진하며 (Pandey et al., 2013; Islam et al., 2014), 또한 salicylic acid 분비를통해스트레스를받는식물의생장을촉진시킨다 (Yusuf et al., 2008). 이외에도 PGPR은식물에서금속결합단백질인 metallothionein 생성을촉진하여금속의독성을감소시키는 (Xia et al., 2012) 등다양한방법으로스트레스하의식물생장을촉진시킬수있다. 그러나이제까지국내에서는금속내성을가진식물생장촉진근권세균에대한연구가많지않았으므로 (Hong et al., 2011), 본연구에서구리내성근권세균을분리하고이들의특성을조사하여구리스트레스를받는식물에내성을부여하며이를이용하여효율적으로식물생장을촉진할수있는가능성을조사하였다. 재료및방법 구리내성균주의분리및특성구리내성세균의분리를위해금, 은, 구리등을채취하던경기도광명시가학광산주변에자생하는식물근권에서토양시료를채취하였다. 시료 1 g을생리식염수 10 ml과혼합하여진탕기 (Recipro shaker RS-1, Jeio Tech) 에서진탕하였다 [300 stroke/m (spm), 30 min]. 구리첨가 Nutrient Broth [NB (Difco Lab) 8 g, CuSO 4 5H 2O 0.3 g, 1 L DW, ph 7] 에토양현탁액을 1% 넣고배양하고 (30 C, 150 rpm, 24 h) 3회계대배양후구리첨가 NB 한천배지에획선배양을반복하여균주를순수분리하였다 (Andreazza et al., 2010). 분리균주의동정을위해 ( 주 ) 마크로젠에 16S rrna 염기서열분석을의뢰하였으며분석된전체염기서열을 National Center for Biotechnology Information (NCBI) 등록균주의염기서열과비교하여상동성을조사하였다. 종수준까지의정확한동정을위해균주의탄소원이용특성을 Biolog EcoPlate TM (Biolog Inc.) 를이용하여조사하였다 (Garland, 1996). 균주를 96 well plate에배양하여 (30 C, 7일 ) 기질이용에따른발색을측정해서 OD 590 이 0.5 이상이면기질이용을양성으로간주하여 + 로나타내고 1.0 이상이면 0.5 간격으로 + 의수를더해서기질이용도를표시하였다. 이결과를염기서열결과와함께이용하여균주를최종동정하였다. 한편선발된균주의구리농도에따른생장을조사하기위해 tryptic soy broth (TSB, Neogen Co.) 에구리의농도가각각 0, 100, 200, 300, 350과 700 mg/l가되도록 CuSO 4 를첨가하여배양하면서 24시간간격으로 OD 600 을측정하였다. 분리균주의구리내성기작균주의구리흡착능조사를위해균주를 NB배지 50 ml에서선배양 (30 C, 150 rpm, 48 h) 하고, 원심분리 (10,000 g, 20 min) 후세포를회수하여멸균증류수로 2번세척하고건조시킨다. 건조된세포 0.1 g을 100 ml의구리용액 (10, 20, 40 mg/l) 에넣어배양 (30 C, 150 rpm) 중시간별로 10 ml씩채취하여원심분리 (3,400 g, 20 min) 후상등액의구리농도를 neocuproine 방법을사용해서측정하였다 (Silva et al., 2009; Ye et al., 2013). 균주의 siderophore 생성능조사는 chrome azurol S (CAS) agar plate assay (Schwyn and Neilands, 1987) 를이용하였는데 CAS 한천평판에구멍을뚫고균주를배양하고 (48 h, 30 C) 주변의 halo를관찰하여 siderophore 생성능을확인하였다. Siderophore의정량은균주를 King s medium B (KB, Difco Lab) 에배양하면서 (48 h, 30 C, 150 rpm) Nagarajkumar 등 (2004) 의방법을이용하여 24시간간격으로측정하였다. 자유라디칼 1,1-diphenyl-2-picryl hydrazyl (DPPH) 제거를통한균주의항산화활성은 NB 50 ml에균주를배양하면서 (30 C, 150 rpm) 24시간간격으로측정하였다. 배양액을원심분리 (8,000 g, 10 min) 하여회수한상등액 0.5 ml을 0.1 mm DPPH 용액 1 ml와증류수 2 ml을함께잘섞고반응 (30 min, 20 C, 암조건 ) 후 517 nm에서흡광도의감소를측정하여항산화활성을다음식으로부터얻었다 (Abo-Elmagd, 2014); Scavenging activity (%) = [A0-(A1-A2)]/A0*100 A0 - 대조군의흡광도값 ( 대조군은배양액대신증류수 ) A1 - 반응혼합물의흡광도값 A2 - DPPH 용액을넣지않은혼합물의흡광도값 (DPPH 대신증류수 ) 분리균주의식물에대한구리내성및생장촉진부여기작 MS1과 MS2 분리균주는 ACC를질소원으로이용하여생장하므로 ACC deaminase 생성능을가진것으로간주되어 Penrose와 Glick (2003) 의방법을이용하여 ACC deaminase 활성을정량하였다. 정량은 α-ketobutyrate를이용하여만든표준곡선을이용하였으며균주의단백질량은 Bradford 방법으로정량하였다 (Bradford, 1976). 균주의살리실산생성능조사는 Zhang 등 (2002) 의방법을이용하였는데 TSB에서균주를배양하면서 (30 C, 150 rpm) 24시간간격으로측정하였다. 배양액을원심분리하여 (3,400 g, 20 min) 상등액을 chloroform으로 2회추출한후원심분리하고 chloroform 층만을회수하여회전농축기로감압증발후 미생물학회지제 53 권제 4 호

식물생장촉진능을가진구리내성세균 253 0.2 μm 공극의여과막으로여과하여 high performance liquid chromatograph (HPLC, Waters Co.) 로분석하였다. 균주의식물호르몬중옥신계열의 indole-3-acetic acid (IAA) 생성은 1 mm의 tryptophan이첨가된 brain heart broth (BHB, Difco Lab) 에서배양 (30 C, 150 rpm) 중 24시간주기로생성능을측정하였다. 배양액을원심분리하여 (3,400 g, 30 min) 상등액을 ethyl acetate로추출하고회수하여감압증발후 methanol을첨가해호르몬을추출하여 HPLC로분석하였다 (Karadeniz et al., 2006). 모든실험은삼반복으로실시하였으며결과는 Student s t-test를통해통계적유의성을조사하였다. 결과및고찰 구리내성균주의분리및특성가학광산주변토양에서구리내성을가지며생장률과 ACC deaminase 활성이가장높은 2개의균주를분리하여 16S rrna 유전자염기서열분석결과 MS1은표준균주인 Pseudomonas veronii CIP104663그리고 MS2는 P. migulae CIP105470과각각 99.0% 의상동성을가지는것으로나타났다. 종수준까지의정확한동정을위해균주의생리생화학적특성조사결과 (Table 1) 각균주는염기서열분석결과와동일한종의탄소이용특성과일치하여 (Palleroni, 2005) 최종적으로 P. veronii MS1과 P. migulae MS2로동정하였다. 구리첨가 TSB배지에서균주들의생장은 300 mg/l까지구리농도증가에따라약간감소하였지만 MS1은 300 mg/l의농도에서도 24시간째 OD 600 이 1.0 이상을, MS2 균주는이보다약간낮은 0.8 이상을나타내며이후에도생장이지속되었는데 (Fig. 1), 이두균주는유사한조건에서 24시간배양시대부분이 0.9 이하의 OD 600 을나타낸 30개의구리광산분리균주들보다구리내성이우수하였다 (Andreazza et al., 2010). 또한구리농도 350 mg/l에서 MS1은배양 48시간에, 700 mg/l에서는 72시간에도 1.0 이상의흡광도가측정되었으며, MS2는 350과 700 mg/l의구리농도에서 96시간배양후에도 1.0 이상의흡광도를나타내어구리존재시내성을가지며높은생장률을지속할수있는것으로나타났다 (Fig. 2). 분리균주의구리내성기작균주의구리흡착능력을조사한결과 MS1과 MS2는 96시간에용액내구리이온 10 mg/l의경우각각 72.6과 69.9%, 20 mg/l의경우각각 64.6과 77.9% 를흡착하였다 (Fig. 3). 이균 주들의세포내구리축적을조사하지는않았지만 20 mg/l 구리수용액에서약 37.5% 의구리이온을흡착및축적으로제거한 Stenotrophomonas maltophilia 보다구리흡착능력이약 2 배이상더높았다 (Ye et al., 2013). 구리농도 40 mg/l에서는 MS1은 48시간에 Cu 2+ 를 27.8% 흡착하고 MS2는 24시간에구리를약 16.7% 흡착한후두균주의구리흡착은더이상증가하지않았다. 구리이온농도에따라두균주의구리흡착능은 Table 1. Metabolic profile of P. veronii MS1 and P. migulae MS2 as tested in the Biolog EcoPlate TM during 5 days Substrate type Substrate P. veronii MS1 P. migulae MS2 Polymer Tween 40 ++ + Tween 80 ++ + α-cyclodextrin - - glycogen + - Carbohydrate D-cellobiose - - α-d-lactose - - β-methyl-d-glucoside - - D-xylose +++ ++ i-erythritol +++ - D-mannitol ++++ ++ N-acety-D-glucosamine ++++ ++ glucose-1-phosphate - - D, L-α-glycerol phosphate - - D-galactonic acid γ-lactone +++ ++ Phenolic 2-hydroxy benzoic acid - - compound 4-hydroxy benzoic acid +++ ++ Carboxylicacid pyruvic acid methyl ester +++ ++ D-galacturonic acid +++ ++ D-glucosaminic acid ++++ ++ γ-hydroxybutyric acid - - itaconic acid +++ - α-ketobutyric acid +++ + D-malic acid ++++ + Amino acid L-arginine +++ ++ L-asparagine ++++ ++ L-phenylalanine + + L-serine +++ ++ L-threonine +++ ++ glycyl-l-glutamic acid +++ ++ Amine phenylethyl-amine + - putrescine ++++ +++ Positive reaction was read if it yielded an OD590 higher than 0.5. -: 0.5 >, +: 0.5~1.0, ++: 1.0~1.5, +++: 1.5~2, ++++: 2.0 < (values: OD 590) Korean Journal of Microbiology, Vol. 53, No. 4

254 Kim and Song Fig. 1. Effect of Cu (II) concentration on growth of P. veronii MS1 (closed bar) and P. migulae MS2 (open bar) in TSB medium amended with Cu (II) after incubation for 24 h. Fig. 3. Residual Cu 2+ after biosorption by P. veronii MS1 ( ) and P. migulae MS2 ( ) in Cu solution (10 (A) and 20 (B) mg/l). Fig. 2. Growth of P. veronii MS1 (A) and P. migulae MS2 (B) in TSB medium amended with 350 (X) and 700 ( ) mg/l of Cu (II). 약간씩달랐지만그다지큰차이는아니었다. 구리를포함한다양한금속과결합하여유리금속농도를크게낮출수있는 siderophore 생성능을조사한결과 MS1 균주는배양 4일째최대 137 ± 41 μmol/ml을생성하였으며 MS2 는 5일째에최대 258 ± 2 μmol/ml을생성하여 (Fig. 4), 12~13 μmol/ml의siderophore 분비량을나타낸근권세균 P. fluorescens PFMDU3, PFMDU8과 PFMDU9보다 10배이상높은생성능을나타냈다 (Nagarajkumar et al., 2004). 모든생물들이금속에노출시유해한활성산소종의형성 Fig. 4. Siderophore production by P. veronii MS1 ( ) and P. migulae MS2 ( ) in King s B medium. 으로이차적인산화스트레스로인해피해를입을수있는데금속내성세균들은다양한방법으로이런산화스트레스를억제할수있다 (Cervantes and Gutierrez-Corona, 1994). 균주의항산화활성조사결과 DPPH 자유라디칼을 24시간에 MS1 은 82.6%, MS2는 78.1% 제거하였고그이후로감소하는경향을보였다 (Fig. 5). 이는유산균인 Lactobacillus bulgaricus CCFM29와 L. casei CCFM30 에서 23.7과 28.84% 의 DPPH 제거 (Zhai et al., 2015) 나진균 Chaetomium madrasense AUMC 미생물학회지제 53 권제 4 호

식물생장촉진능을가진구리내성세균 255 Fig. 5. Scavenging activity of 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radicals by P. veronii MS1 ( ) and P. migulae MS2 ( ) in LB medium. 9376의최대 70.21% 제거 (Abo-Elmagd, 2014) 보다훨씬높은항산화활성이었다. 분리균주의식물에대한구리내성및생장촉진부여기작 분리균주가단일질소원으로 ACC를이용하여생장할수있는것을확인하여 ACC deaminase 생성능이있는것으로판단하였으며, 스트레스에틸렌의전구체인 ACC를 α-ketobutyrate 와 ammonia로가수분해하는 ACC deaminase 활성을정량한결과 4일째에 MS1과 MS2는각각 7.10 ± 0.05와 6.42 ± 0.01 μmol α-ketobutyrate mg/h의가장높은활성을보였다. 이는중금속스트레스하에서식물의뿌리길이생장을촉진한다고보고된 P. fluorescens ACC9의 ACC deaminase 활성인 1.57 ± 0.12 μmol α-ketobutyrate mg/h와 P. tolaasii ACC23의 1.16 ± 0.09 μmol α-ketobutyrate mg/h보다더높았으며 (Dell Amico et al., 2008), 또한 ACC deaminase를분비하는 5개의 Rhizobium 균주들의 0.43~1.78 μmol α-ketobutyrate mg/h와비교할때 6~10배더높은활성이었다 (Ma et al., 2003). 대표적인식물생장촉진호르몬인 IAA는다양한 PGPR이분비하는데 MS1과 MS2는모두 48시간째각각최대 42.9 ± 2.2와 60.8 ± 3.9 μm을분비하였다. 이는다른 IAA 생성 PGPR 에비해소량이지만 IAA는아주미량으로도식물생장을촉진시킨다고알려져있으며실제식물체내에서도 IAA는낮은농도로존재하며오히려고농도의 IAA는뿌리생장을억제하고에틸렌생성을촉진한다고한다 (Pilet and Saugy, 1987). 한편 ACC deaminase와 IAA를모두분비하는세균을처리한식물체내의에틸렌수준이 IAA만분비하는세균을접종한식물만큼증가되지않는데그이유는 ACC deaminase의활성때문에에틸렌생성이저해되며그결과에틸렌피드백에의한 IAA 신호전달이저해되지않아결과적으로식물에틸렌수준은낮추면서생성된 IAA에의해식물생장을자극할수있다 (Glick Fig. 6. Salicylic acid production by P. veronii MS1 ( ) and P. migulae MS2 ( ) in TSB medium. et al., 1998). 따라서 ACC deaminase와 IAA를모두분비하는 MS1과 MS2는구리스트레스하의식물생장도효율적으로증가시킬수있을것이다. MS1과 MS2는 salicylic acid를 4일째에각각최대 17.42 와 28.33 μg/ml을생성하였는데 (Fig. 6), Serratia marcescens 90-166 균주가분비한 38.6 ng/ml보다훨씬더높은수준이다. 식물이스트레스를받지않을때는 salicylic acid의분비가별다른효과가없지만비생물적스트레스를받는식물에 0.1 μm 의 salicylic acid를처리했을때식물의생장, 광합성, 엽록소양이증가했다고보고되었다 (Yusuf et al., 2008). 이상의결과를종합해볼때본연구에서분리한 P. veronii MS1과 P. migulae MS2는구리에내성을가지며식물생장촉진특성을가지고있기때문에구리를포함하여금속스트레스를받는식물에처리할경우식물생장을촉진하여금속오염환경의식생복원및금속오염정화에활용할수있을것으로예상된다. 적요 구리스트레스를받는식물에구리내성을부여하며생장을촉진할수있는여러구리내성근권세균을분리하였다. 분리균주 Pseudomonas veronii MS1과 P. migulae MS2는금속킬레이트제인 siderophore를각각 0.13과 0.26 mmol/ml 생성하였으며또한 20 mg/l Cu 수용액에서각각 64.6과 77.9% 의구리에대한생물흡착능을나타내었다. 구리는생물체에서산화스트레스를유발할수있는유해한자유라디칼형성을유도할수있다. MS1과 MS2 균주의 1,1-diphenyl-2-picryl hydrazyl 라디칼제거능과항산화능은 24시간배양후대조군에비해각각 82.6과 78.1% 증가하였다. 이들은식물에서스트레스호르몬 Korean Journal of Microbiology, Vol. 53, No. 4

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