Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Cellular Responses of the TNT-degrading Bacterium, Stenotrophomonas sp. OK-5 to Explosive 2,4,6-Trinitrotoluene (TNT)

폭약 2,4,6-Trinitrotoluene에 노출된 분해세균 Stenotrophomonas sp. OK-5의 세포반응

  • 장효원 (순천향대학교 생명과학부) ;
  • 송승열 (순천향대학교 생명과학부) ;
  • 김승일 (기초과학연구원 프로테옴분석팀) ;
  • 강형일 (순천대학교 환경교육과) ;
  • 오계헌* (순천향대학교 생명과학부)
  • Published : 2002.12.01
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Abstract

The cellular responses of TNT-degrading bacterium, Stenotrophomonas sp. OK-5 to explosive 2,4,6-trini-trotoluene (TNT) as an environmental contaminant were examined. Survival of the strain OK-5 with time in the presence of different concentrations of TNT under sublethal conditions was monitored, and viable counts paralleled the production of the stress shock proteins in this bacterium. Total cellular fatty acids analysis showed that strain OK-5 produced or disappeared several different kinds of lipids when grown on TNT media than when grown on TSA. Under scanning electron microscope, the cells treated with 0.5 mM TNT for 12 hrs showed irregular rod shapes with wrinkled surfaces. Analyses of SDS-PAGE and Western blot using anti-DnaK and anti-GroEL revealed that several stress shock proteins including 70 kDa DnaK and 60 kDa GroEL in strain OK-5 were newly synthesized at different TNT concentrations in exponentially growing cultures. 2-D PAGE of soluble protein fractions from the culture of OK-5 exposed to TNT demonstrated that approximately 300 spots were observed on the silver stained gel ranging from pH 3 to pH 10. Among them, 10 spots significantly induced and expressed in response to TNT were selected and analyzed. As the result of internal amino acid sequencing with ESI-Q TOF, two proteins, spot #1 and spot #10 were assigned the DnaK protein XF2340 of Xylella fastidiosa and stress-induced protein of Mesorhizobium loti, respectively.

환경오염원으로서 폭약 2,4,6-trinitrotoluene (TNT)에 대한 TNT 분해세균 Stenotrophomonas sp. OK-5의 세포반응에 대하여 조사하였다. 아치사조건의 TNT농도와 노출시간에 따른 균주 OK-5의 생존율을 분석한 결과, 이 세균의 생존율은 스트레스 충격 단백질의 생성과 비례하였다. 총세포 지방산 조성분석에서 균주 OK-5는 tryp-ticase soy agar에서 자랄 때보다 TNT 배지에서 자랄 때 여러 가지 종류의 지방산이 생성되거나 사라지는 것이 밝혀졌다. 주사전자현미경하에서 TNT에 노출된 세포는 쭈글쭈글하고 불규칙적인 간상형으로 나타났다. Anti-DnaK와 anti-GroEL을 이용하여 SDS-PAGE와 Western blot을 통한 분석으로 균주 OK-5는 70 kDa DanK와 60 kDa GroEL을 포함하는 몇가지 스트레스충격단백질을 생성하는 것으로 밝혀졌다. TNT에 노출된 OK-5 배양에서 수용성 단백질 분획에 대하여 2-D PAGE를 실시하였으며, pH 3에서 pH 10의 범위에서 약 300여 개 spot들이 silver로 염색된 gel상에서 관찰되었다. 이들 가운데 TNT의 반응으로 현저하게 유도되고 발현된 10개의 spot들을 확인하였으며, 2개의 단백질, spot #1과 spot #10에 대한 내부아미노산 서열을 ESI-Q TOF로 분석한 결과, Xylella fastidiosa의 DnaK protein XF2340와 Mesorhizobium loti의 스트레스 유도단백질로 각각 밝혀졌다.

Keywords

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