Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Characterization of a PyrR-deficient Mutant of Bacillus subtilis by a Proteomic Approach

프로테옴 분석에 의한 Bacillus subtilis PyrR 돌연변이체의 특성

  • Seul, Keyung-Jo (School of Life Sciences, Kyungpook National University) ;
  • Cho, Hyun-Soo (Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo) ;
  • Ghim, Sa-Youl (School of Life Sciences, Kyungpook National University)
  • 설경조 (경북대학교 생명과학부) ;
  • 조현수 (동경대학 의과대학 세포생물학 및 해부학과) ;
  • 김사열 (경북대학교 생명과학부)
  • Received : 2010.05.25
  • Accepted : 2010.11.29
  • Published : 2011.03.28
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Abstract

The Bacillus subtilis pyrimidine biosynthetic (pyr) operon encodes all of the enzymes for the de novo biosynthesis of Uridine monophosphate (UMP) and additional cistrones encoding a uracil permease and the regulatory protein PyrR. The PyrR is a bifunctional protein with pyr mRNA-binding regulatory funtion and uracil phosphoribosyltransferase activity. To study the global regulation by the pyrR deletion, the proteome comparison between Bacillus subtilis DB104 and Bacillus subtilis DB104 ${\Delta}$pyrR in the minimal medium without pyrimidines was employed. Proteome analysis of the cytosolic proteins from both strains by 2D-gel electrophoresis showed the variations in levels of protein expression. On the silver stained 2D-gel with an isoelectric point (pI) between 4 and 10, about 1,300 spots were detected and 172 spots showed quantitative variations in which 42 high quantitatively variant proteins were identified. The results showed that production of the pyrimidine biosynthetic enzymes (PyrAA, PyrAB, PyrB, PyrC, PyrD, and PyrF) were significantly increased in B. subtilis DB104 ${\Delta}$pyrR. Besides, proteins associated carbohydrate metabolism, elongation protein synthesis, metabolism of cofactors and vitamins, motility, tRNA synthetase, catalase, ATP-binding protein, and cell division protein FtsZ were overproduced in the PyrR-deficient mutant. Based on analytic results, the PyrR might be involved a number of other metabolisms or various phenomena in the bacterial cell besides the pyrimidine biosynthesis.

Bacillus subtilis의 pyrimidine biosynthetic (pyr) operon은 UMP의 de nove 생합성에 관여하는 enzyme들을 encode할 뿐만 아니라, 조절단백질인 PyrR도 encode한다. PyrR은 pyr mRNA-binding 조절 기능과 uracil phosphoribosyltransferase activity를 동시에 가지는 bifunctional 단백질이다. 본 연구에서는 Proteomic analysis를 이용하여 Uracil - 환경에서 DB104${\Delta}$pyrR의 단백질 패턴을 분석하여 단백질 레벨에서 PyrR 단백질의 실질적인 조절 양상을 관찰하였다. 두 균주의 세포질 단백질은 다양한 발현의 차이를 보였으며, Silver 염색된 2D-gel의 pI 4~10 사이에서는 1,300여개의 단백질이 검출되었으며, 단백질 발현 차이를 보이는 172개의 spot 중에서 42개의 단백질이 identification 되었다. 그 결과 pyr operon의 단백질(PyrAa, PyrAb, PyrB, PyrC, PyrD, and PyrF)이 모두 Up regulation이 이루어지고 있음을 확인할 수 있었으며, 이것은 단백질 레벨에서 Pyrimidine 생합성 과정이 PyrR에 의해서 정확히 Regulation 되어짐을 확인할 수 있었다. 또한 Pyrimidine 생합성의 Up regulation과 Down regulation 상태의 단백질의 패턴 양상도 분석할 수 있게 되었다. Pyrimidine의 생합성 과정은 DNA를 구성하는 기본적인 구성 요소를 생산하는 과정으로서 여러가지 Metabolism 가운데 중요한 위치를 차지하고 있다. 만약 Pyrimidine의 생합성 과정이 Over- expression된다면 다른 Metabolism의 균형에도 변화가 올 것이다. Proteomics Analysis에 이용한 DB104${\Delta}$pyrR 균주는 Pyrimidine 생합성의 조절에 관여하는 PyrR knock out 균주로서 Uracil - 환경에서는 전체적인 Pyrimidine 생합성 조절이 Up regulation이 되어지므로 Up regulation 동안 어떤 Metabolism에 영향을 주는지 관찰을 할 수 있게 되었다. 특히 Amino Acid Metabolism에 관계있는 단백질의 Up regulation이 이루어짐을 관찰할 수 있었으며 이것은 현재 각광을 받고 있는 단백질 산업에 응용함으로써 산업적으로 많은 기대를 할 수 있을 것으로 예상되어진다.

Keywords

References

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