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Characteristics of Heavy Metal Resistant Plasmid in Enterobacter cloaceae K41

Enterobacter cloaceae K41 plasmid의 중금속 저항성

  • Kim Young-Hee (Dept. of Life Science and Biotechnology, Dong-Eui University) ;
  • Lee Sang-Jun (Dept. of Microbiology, Pusan National University) ;
  • Jeong Yong-Kee (Dept. of Life Science and Biotechnology, Dong-Eui University) ;
  • Chung Kyung-Tae (Dept. of Life Science and Biotechnology, Dong-Eui University)
  • 김영희 (동의대학교 생명응용과학과) ;
  • 이상준 (부산대학교 미생물학과) ;
  • 정영기 (동의대학교 생명응용과학과) ;
  • 정경태 (동의대학교 생명응용과학과)
  • Published : 2005.08.01

Abstract

A natural habitat bacterium, Enterobacter cloaceae K41 was isolated from fresh water plant root and identified. This strain was used to investigate heavy metal resistance. The optimal growth conditions of the bacterium were LB medium containing$1\%$ yeast extract, $1\%$ lactose, $1\%$ NaCl, pH 7.0, at $37^{\circ}C$, and for 24 hours on a shaker. The minimal inhibitory concentration (MIC) of heavy metals against E. cloaceae KCTC2519 and E. cloaceae K41 was compared. The MIC of E. cloaceae K41 was 150 ppm in Cu, 50 ppm in Cd whereas that of the standard strain was 50 ppm in Cu but no growth was observed either Cd or two mixed heavy metal solution. The presence of plasmid was cleared from the isolated strain whereas no possession from the standard strain. The plasmid from E. cloaceae K41 was transformed into E. coli $DH5{\alpha}$. The MIC of transformed strain increased resistance 7 times in Cu and 6 times in Cd by insertion of this plasmid. The metal adsorption of the transformant was increased 1.3 times in Cu and 1.5 times in Cd indicating the plasmid was responsible for heavy metal resistance.

담수 식물 수초의 근계에 부착하는 미생물 중 중금속에 높은 저항성을 가지는 균을 분리하고 이들 중 Enterobacter cloaceae K41를 대상으로 생육최적조건을 검토한 결과는 LB배지에 $1\%$ yeast extract, $1\%$ lactose, $1\%$ NaCl, pH7.0, 최적 온도는 $37^{\circ}C$, 24시간 진탕배양 이었으며 중금속 첨가 시에는 침전을 막기 위하여 Nutrient 배지로 대체하였다. 이 분리균주와 표준균주인 Enterobacter cloaceae KCTC2519를 대상으로 중금속인 구리와 카드뮴이온에 대한 최소생육저지농도(MIC)를 비교한 결과 분리균주인 E. cloacear K41은 구리는 150 ppm, 카드뮴은 50 ppm농도까지 생육이 확인되었으나 표준균주는 구리 50ppm에서 생육이 확인되었으나 카르뮴이나 두 혼합 중금속에서는 확인되지 않는 차이를 나타내었다. 두 균주를 대상으로 유전적 성상을 비교한 결과 분리균주에선 plasmid가 검출되었으나 표준균주에는 없었다. 그리고 분리균주에서 6.4Kb 절편의 plasmid를 분리하여 구리, 카드뮴의 중금속에 민감한 균주인 E, coli $DH5{\alpha}$에 형질전환 시켜 생육에 영향을 미치는 정도를 비교하였다. 그 결과 형질전환 균주의 두 중금속에 대 한 최소생육저지농도가 구리는 7배, 카드뮴은 6배로 증가한 것을 알 수 있었다. 또한 중금속 흡착률은 형질전환 균주가 E. ccli $DH5{\alpha}$보다 구리가 1.3배, 카드뮴은 1.5배 증가함을 알 수 있었다. 따라서 이 plasmid가 중금속 저항성을 증가시키는데 관여하는 것으로 보였다.

Keywords

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