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1 Curr. Top. LAB Probiotics Vol. 1, No. 1, pp. 50~54(2013) Streptococcus thermophilus 의자연적형질전환메커니즘과 Leuconostoc 속세균에서그메커니즘의추론 박재용 * 대구가톨릭대학교식품영양학과 Natural Transformation of Streptococcus thermophilus and the Possibility of Identifying the Mechanism in the Genus Leuconostoc Jae-Yong Park* Department of Food Science and Nutrition, Catholic University of Daegu, Gyengsan , Korea Abstract: Natural genetic transformation is one of mechanism for horizontal transfer of genetic elements in bacteria which is an integral part of the physiology of bacteria. Streptococcus thermophilus utilize a novel quorum sensing system to control for development of natural competence which composes Rgg-type response regulator (ComR) and hydrophobic peptide pheromone (ComS). Matured ComS is released outside of cell during early exponential phase when growing in peptide free chemical defined medium (CDM). Matured ComS is transported via Ami (oligopeptide transport system) form outside to cytoplasm, and then bind to ComR. Activated ComR (matured ComS combining form of ComR) induces transcription of comx, which encodes an alternative sigma factor (σ x ) that regulates expression of late competence genes. Adding synthetic matured form of ComS to CDM, it can induce genetic transformation of S. thermophilus. The genus Leuconostoc has not been identified natural transformation mechanism even though presenting homologous genes related with late competence development. The study for identifying natural transformation mechanism of Leuconostoc can realize development of improved strains for kimchi industry. Keywords: natural genetic transformation, Streptococcus thermophilus, Leuconostoc, kimchi 서 한국의전통발효식품인김치는살균과정없이발효를진행하기때문에, 원재료에서유래된여러가지다양한미생물들이발효에관여하게되며, 최근의특이이들중주종을이루는것은 Leuconostoc, Lactobacillus, Weissella 속의여러세균들이다 (Cho et al., 2006; Jung et al., 2011; Park et al., 2012). 그중에서도특히배추김치의발효과정에서가장중요한역할을하는미생물중하나는 Leuconostoc mesenteroides를비롯한 Leuconostoc 속세균들로알려져있으며 (Jung et al., 2011), Leu. mesenteroides를스타터로 *Corresponding author: Jae-Yong Park, Department of Food Science and Nutrition, Catholic University of Daegu, Gyengsan , Korea. Tel: , Fax: jaepark@cu.ac.kr Received October 11, 2012; Revised November 29, 2012; Accepted December 14, 2012 론 사용하였을때 Leuconostoc 속세균이우점종을이루게되어김치내의유리당을보다빠르게소비하여젖산과만니톨을생산하는것으로확인되었다 (Jung et al., 2012). 세계적인건강발효식품으로서김치를세계화하기위해서는고품질의김치를일정한품질로유지하면서대량생산및유통할수있는시스템의구축이반드시필요하게되었다. 우수한품질이일정하게유지되는김치를대량생산하기위해서는, 김치발효에서중요한역할을하는김치유산균들, 특히 Leuconostoc 속유산균들의생리, 대사등에대한깊이있는이해가필수적이다. 이를위해서는아직까지그기능이밝혀지지않은유전자들이암호화하고있는단백질들의기능을밝히는연구가필요하다. 현재까지전체염기서열이밝혀진 Leu. mesenteroides (Jung, et al., 2012; Makarova, et al., 2006), Leu. citreum (Kim et al., 2008), Leu. kimchii (Oh et al., 2010), Leu. inhae (Kim et al., 2010) 에서기존에많은연구가된 Bacillus subtilis나 Lactococcus lactis와같은 GC 함량이낮은모델그램양성세균들과의비교유전체학을통해서많은유전자들의기능이 50

2 Natural Transformation of Streptococcus thermophillus and Identifying Mechanism in Leuconostoc 51 예측되고있으나, 예측된기능을실제로그유전자가담당하고있는지에대한검증에현실적인어려움이있다. 또한아직까지도그기능을예측하지못하고있는유전자들의기능을밝혀야하는연구도산재되어있다는점에서 Leuconostoc 속유산균들의생리와대사를이해하는데는많은연구가필요하다. 이러한유전자의기능적연구를위해서해당유전자의기능을없애는유전자 knock-out 기술이수반되어야한다. 몇몇연구팀에서 Leuconostoc 속유산균에서적용할수있는유전자 knock-out 기술개발을위해서많은시도들을하였으나, 현재까지성공한사례가없다. 유산균과같은 Firmicutes에속하는 B. subtilis의경우, 상대적으로매우쉽게목적유전자를 knock-out시킬수있는데, 이는자연적으로일어나는형질전환 (natural genetic transformation) 에기초한 knock-out 방법을사용하기때문이다. 이러한형질전환현상은 Streptococcus pneumoniae 를이용한 Grittith의유명한실험에서최초로발견된현상이다 (Griffith, 1928). 지금현재까지이러한자연적인형질전환현상이보고된세균은 60종이상이며, 이들세균들은기본적으로형질전환이가능한상태 (competence) 의세포내에서만발현되는 DNA 수송단백질단 (type IV pili와 type II 분비시스템 ) 을보유하고있다 (Chen and Dubnau, 2004). DNA를세포내로수송하고상동유전자재조합 (homologous recombination) 이일어나는메커니즘은 60여종의세균에서거의비슷하나, competence 세포로유도되는과정은매우다양하여, 진화적으로매우가까운종간에서만비슷한메커니즘을사용하는것으로보인다. 최근들어낙농유산균인 Streptococcus thermophilus에서형질전환이가능한상태로유도되는현상이발견되면서 (Gardan et al., 2009), 유산균에서도자연적형질전환현상을이용한 knock-out 방법개발의가능성이확인되었다. 본총설에서는 S. thermophilus 의형질전환초기반응 (competence가유도되는반응 ) 의기작을정리하고, 김치유산균인 Leuconostoc 속유산균의후기형질전환유전자들 (DNA 수송과상동유전자재조합관련유전자들 ) 의존재에대해서정리할것이다. S. thermophilus의자연적형질전환현상자연적인형질전환현상이가장먼저발견된 S. pneumoniae와같은속에속하지만유산균인 S. thermophilus의자연적형질전환현상은최근까지확인되지않고있었다. S. thermophilus의자연적인형질전환현상의메커니즘을푸는데첫번째단추는프랑스의 Monnet 그룹에서올리고펩타이드수송시스템 (oligopeptide transport systems) 인 Ami 에대한연구과정에서풀어지게되었다. Ami는그램양성세균에서세포내 외부로올리고펩타이드를수송하는역할을담당하는데이는질소원으로서의올리고펩타이드의흡수하는역할뿐만아니라, 펩타이드페르몬 (peptide pheromone) 을세포내로전달함으로써세균의세포간상호의사소통 (cell-to-cell communication) 방식인 quorum sensing 에서중요한부분을담당하고있는것으로알려져있다 (Waters and Bassler, 2005). Monnet 그룹은 S. thermophilus에서 Ami를통한 quorum sensing이일어나지는지그리고이 quorum sensing에의해세포내어떤변화가생기는지에관심이있었다. 이들은 Ami의 C, D, E 세개의 subunit가 knock-out된변이주를 peptide가없는화학적제한배지 (chemically-defined medium, CDM) 에서배양하면서야생형균주와발현되는단백질의변화를프로테옴분석을통해서확인하였는데, 야생형에서는발현되다가 ami 변이주에서는전혀생성되지않는 17개의단백질중 7개가 S. pneumoniae의후기형질전환단백질과서열이유사한단백질이었다 (Gardan et al., 2009). S. thermophilus 배양에주로사용하는 M17 배지에서는형질전환이가능한상태로변화가되지않지만, CDM에서는대수기초기에짧은시간동안형질전환이가능한상태로변화된다는것또한확인되었고, Ami 단백질중올리고펩타이드결합단백질인 AmiA3가형질전환능력을가지게하는데아주중요하다는사실또한확인되었다 (Gardan et al., 2009). S. thermophilus 의자연적형질전환유도메커니즘 Monnet 그룹의중요한발견을기초로, Hols 그룹에서 S. thermophilus의초기자연적형질전환능력생성의메커니즘이거의밝혀지게되는데 (Fontaine et al., 2010), 이들은 CDM에서 Streptococcus 속세균의형질전환유전자들의생성의가장중요한열쇠인형질전환유도 sigma factor를암호화하는 comx의발현이증가하는비슷한시기에발현이극적으로증가하는새로운유전자를발굴하기위해서 microarray를이용하였다. Ami 결핍변이주와 ComX 결핍변이주사이에서발현의변화가 100배이상차이가나는 4 개의유전자군이발견되었으며, 이들중하나는뒤에 Rgg 패밀리에속하는조절단백질을포함하고있었고이단백질은나중에 ComR로명명되었다. 나머지 3개의유전자군은각각박테리오신유전자를포함하고있었는데, 이는자연적형질전환능력을갖고있는여러다른 Streptococcus 속세균들에서형질전환능력유도와박테리오신의생산은동시에일어나고, 이는외부의 DNA를받아들이는데유용하다는것이알려져있다 (Claverys et al., 2007; Perry et al., 2009). comr 유전자와이웃한 24개의아미노산을구성된소수성펩타이드를암호화하는유전자 ( 뒤에 coms로명명 ) 를 knock-out시킨경우 comx의발현이전혀일어나지않는것이확인되어, coms와 comr이형질전환이가능한상태로되는데필수적인유전자임이확인되었다. 또한 CDM 에서 coms의변이주를배양하면서합성된 ComS의 C-말

3 52 Curr. Top. LAB Probiotics, Vol. 1, No. 1 (2013) 단서열을가진펩타이드를첨가한경우형질전환능력이회복되는것으로나타나, coms 유전자가 quorum sensing의펩타이드페르몬을암호화하는것으로확인되었다. 또한 ComS의 C-말단서열을가진펩타이드는형질전환능력을가지지못하거나아주약한다른 S. thermophilus 균주와 Streptococcus salivarious 균주의형질전환능력을유도하는것으로확인되었다. 따라서기존의형질전환능력이확인되었던 mitis나 mutans 그룹의 Streptococci들은 histidine kinase, response regulator, 펩타이드페르몬으로구성된 threecomponent 시스템에의해서형질전환능력이유도되는반면 salivarious 그룹은 histidine kinase를가지지않은 ComRS two-component 시스템에의해서형질전환능력이유도된다 (Fig. 1)(Fontaine et al., 2010). 그리고분자샤페론의일종인 ClpC와여기에결합할수있는 MecA는 ComX를분해시켜형질전환능력을보유한상태를원래대로되돌리는역할을한다 (Fig. 1)(Biørnstad and Hvarstein, 2011; Boutry et al., 2012). S. thermophilus 의자연적형질전환의응용 S. thermophilus의형질전환유도메커니즘을활용하여산업적으로활용할수있는새로운균주를개발하려는노력이시도되고있는데, 그첫시도는우유에서빠르게산을생성할수있는변이주개발에관한것이다. ComS의 C- 말단서열을가진펩타이드를 CDM에서처리하여 cellenvelope protease를암호화하는 prts 유전자부위를염색체상에항생제내성유전자마커없이삽입시킴으로써우유에서빠르게산을생성할수있는균주로개량하는데성공하였다 (Dandoy et al., 2011). 이러한예는유산균에서자연적형질전환유도메커니즘을밝히는것이학문적으로유산균의유전및생리적인성질을이해하는데도도움을 주지만, 실제산업적으로활용가능한균주개발에도활용될수있다는사실을보여준다. Leuconostoc 속유산균의자연적형절전환능력보유가능성 김치유산균인 Leuconostoc 속세균에서유전자의인위적인 knock-out에성공한사례는현재까지없지만자연적인형질전환현상이관찰된보고는있다. Leuconostoc carnosume 에서자연적형질전환 electrophoration을위해준비된세포를 plasmid DNA와섞어두기만해도형질전환이일어나는것이보고된바있으나그메커니즘은여전히베일에가려져있는상태이다 (Helmark et al., 2004). 다른속의 Streptococci들에서관찰되었던자연적형질전환현상이최근들어서 S. thermophilus에서발견된사실을통해서알수있듯이, Leu. carnosume에서발견된것과같이다른 Leuconostoc 속유산균에서도자연적형질전환현상이발견될가능성은아직열려있다. 또한김치유산균이자연적으로생육하는환경인김치에서는제한된영양원을획득하기위해서다양한세균들과경쟁해야하는상황이며, 최근들어보고되고있는김치유산균에치명적인박테리오파아지에도노출되고있는환경이기때문에이를극복하기위해서외래 DNA를적극적으로받아들여야할필요성이있다. 전체염기서열이결정된 Leu. mesenteroides와 Leu. citreum의유전체를분석해보면, B. subtilis를비롯한자연적형질전환현상이보고된그램양성세균의후기형질전환유전자들과아미노산서열에서유사성을갖는후기형질전환유전자로추정되는유전자들을다수발견할수있다 (Table 1). 그러나 B. subtilis의초기형질전환유도와관련된유전자들과서열의유사성을갖는유전자를 Leu- Fig. 1. Schematic representation of early competence regulation in S. thermophilius. Genes and its encoding proteins are connected with solid line arrows. Dashed line arrows indicate activation of gene expression.

4 Natural Transformation of Streptococcus thermophillus and Identifying Mechanism in Leuconostoc 53 Table 1. List of homologous genes of Leuconostoc with late competence genes from B. subtilis Loci Gene Accession # Annotated function Leu. mesenteroides Leu. citreum ATCC8293 KM20 comc comc NP_ membrane protease and transmethylase - YP_ comer NP_ late competence protein ComER - YP_ come comea NP_ membrane bound high affinity DNA binding receptor YP_ YP_ comeb NP_ Putative enzyme associated to DNA transport YP_ YP_ comec NP_ DNA channel for uptake in competent cell YP_ YP_ comfa NP_ helicase competence protein YP_ YP_ comf comfb NP_ hypothetical protein BSU comfc NP_ putative component of the DNA transport apparatus YP_ YP_ comga NP_ membrane associated factor for DNA competence YP_ YP_ comgb NP_ membrane platform component of DNA transport machinery YP_ YP_ comgc NP_ pilin-like component of DNA transport membrane platform YP_ YP_ comg comgd NP_ membrane component of DNA transport platform YP_ YP_ comge NP_ component of DNA transport platform - YP_ comgf NP_ component of DNA transport platform YP_ comgg NP_ component of DNA transport platform - - reca reca NP_ recombinase A YP_ YP_ addab adda NP_ ATP-dependent deoxyribonuclease (subunit A) YP_ YP_ addb NP_ ATP-dependent deoxyribonuclease (subunit B) YP_ YP_ comk comk NP_ competence transcription factor (CTF) - - conostoc 속유산균들은보유하고있지않은데, Leuconostoc 속유산균들은또다른방식의초기형질전환유전자발현시스템을보유하고있는것으로보인다. 또한 streptococci 들의초기형질전환반응의핵심적인역할을하는 threecomponent 시스템의 histidine kinase와 response regulator 들과서열의유사성을갖는유전자들은발견되지만, 이는세균에서널리존재하는여러가지종류의 histidine kinase 와 response regulator들이서열의유사성을가지기때문에초기형질전환반응을유도할가능성은미지수다. 따라서 Leu. mesenteroides와 Leu. citreum과같은김치유산균이외래 DNA를세포내로유입시켜상동유전자재조합이일어나는데필요한유전자들은보유하고있으나, 이들유전자들의발현을유도할수있는조건과이를매개하는메커니즘이밝혀지지않은상태라할수있다. 요약자연적으로일어나는형질전환은세균에서유전요소의수평적전달메커니즘의하나로서세균생리학에서빠질수없을만큼중요한현상이다. S. thermophilus는자연적으로형질전환이가능한상태로변환하기위해서 Rgg-타입의 response regulator인 ComR과소수성펩타이드페르몬인 ComS를포함하는새로운 quorum sencing 시스템을이용한다. S. thermophilus를화학적제한배지에서배양하면대수기초기에신호서열이분리된 ComS는세포외부로배출된다. 배출된 ComS는올리고펩타이드전달시스템인 Ami를통하여세포내로유입되어 ComR과결합하게된다. ComS와결합된활성화된 ComR은후기형질전환관련유 전자들의전사를조절하는대체형시그마펙터 (σ x ) 를암호화하는 comx 유전자의발현을유도함으로써형질전환이일어나도록만든다. 신호서열이제거된 ComS를합성하여화학적합성배지에첨가하게되면, S. thermophilus의형질전환을인공적으로유도할수있다. Leuconostoc 속유산균들은그들이후기형질전환관련유전자들과서열이유사한단백질유전자들을보유하고있음에도불구하고자연적형질전환메커니즘은아직밝혀지지않았다. Leuconostoc 속의자연적형질전환의메커니즘을규명하는연구는김치산업에서사용될수있는개량된균주개발을가능하게할것이다. 참고문헌 Biørnstad TJ and Hvarstein LS (2011) ClpC acts as a negative regulator of competence in Streptococcus thermophilus. Microbiol. 157, Boutry C, Wahl A, Delplace B, Clippe A, Fontaine L, and Hols P (2012) Adaptor protein MecA is a negative regulator of the expression of late competence genes in Streptococcus thermophilus. J. Bacteriol. 194, Chen I and Dubnau D (2004) DNA uptake during bacterial transformation. Nat. Rev. Microbiol. 2, Cho J, Lee D, Yang C, Jeon J, Kim J, and Han H (2006) Microbial population dynamics of kimchi, a fermented cabbage product. FEMS Microbiol. Lett. 257, Claverys JP, Martin B, and Hvarstein LS (2007) Competenceinduced fratricide in streptococci. Mol. Microbiol. 64, Dandoy D, Fremaux C, Frahan MHd, Horvath P, Boyava P, Hols P, and Fontainel L (2011) The fast milk acidifying phenotype

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untitled 3. 농업환경연구과 과제구분 기본연구 수행시기 전반기 연구과제 및 세부과제 수행 기간 소 속 책임자 농가에 적합한 부식성곤충 대량 사육기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 1) 부식성 곤충 먹이 제조 기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 색인용어 부식성곤충, 장수풍뎅이, 계통, 먹이제조 ABSTRACT In first check,