S-Space College of Natural Sciences (자연과학대학) Dept. of Biological Sciences (생명과학부) Theses (Ph.D. / Sc.D._생명과학부)
Relationship between polyamine and methylglyoxalin the cell elongation of Bacillus subtilis
Bacillus subtilis에서 세포 신장에 관여하는 polyamine과 methylglyoxal의 관계
- 자연과학대학 생명과학부
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2015. 8. 강사욱.
- Methylglyoxal is the metabolic intermediate and acts as a growth- or division inhibitor in cells. This ?-ketoaldehyde, which have been recognized as the advanced glycation end product precursors such as glyoxal, methylglyoxal and deoxyglucosone, have been suggested to be regulated by endogenous or exogenous polyamines. Polyamines, which are abundant compounds in cells from bacteria to mammals, generally participate in various cell physiology such as the cell growth, division and differentiation.
Herein, the methylglyoxal was observed to react with several types of polyamines in vitro resulting from the electron paramagnetic resonance spectroscopy. To investigate the physiological function and relationship of both methylglyoxal and polyamines in Bacillus subtilis, polyamine biosynthesizing-genes such as speA, speB, and yaaO encoding the arginine decarboxylase, agmatinase, and lysine decarboxylase, were disrupted or overexpressed respectively. The growth rate was slightly retarded in all of the polyamine-deficient strains compared to wild-type cells. Additionally, the significant growth defect was observed in the speBOE and yaaOOE strains not in the case of the speAOE strain. The intracellular methylglyoxal content increased 2.5- to 3-fold in the speBOE and yaaOOE strains compared to the reference strain. In addition, the mgsA gene expression, which is supposed to encode methylglyoxal synthase based on the genome database of B. subtilils, increased in the speBOE and yaaOOE strains. Based on these observations of the changes in the cell physiology in these cells, we microscopically examined whether the effect of the increased methylglyoxal or polyamine content might affect the cellular morphology. Over 60% of the elongated cells were observed in the speBOE and yaaOOE strains contrast to the reference strain. The expression of actin-like mreB, which codes a protein involved in the cell elongation predominantly in prokaryotes, increased in the speBOE and yaaOOE strains. These results indicated that methylglyoxal accumulation by the mgsA gene expression in the speBOE and yaaOOE strains caused the growth inhibition and altered the cell morphology by triggering mreB expression.
To reveal the relationship between cellular methylglyoxal and cell shape formation under other experimental conditions, Bacillus methylglyoxal synthase was purified using the Escherichia coli recombinant protein fused with the pET3a vector system. The Km and kcat values for Bacillus methylglyoxal synthase activity were revealed to be 3.17 mM and 0.009 (s-1) respectively, when using dihydroxyacetone phosphate as a major substrate. The optimal temperature and pH for the Bacillus methylglyoxal synthase activity were observed 40?C and 5.5, respectively and furthermore native molecular mass was calculated as a dimeric form. Bacillus methylglyoxal synthase activity decreased in an exogenous phosphate compounds concentration-dependent manner. These results indicated that Bacillus methylglyoxal synthase encoded by the mgsA gene catalyze dihydroxyacetone phosphate into methylglyoxal and showed a different biochemical properties compared to other bacterial proteins.
Interestingly, the mgsAOE cells showed a drastic increase in the cellular methylglyoxal content ranging from 2.5- to 3.5-fold higher compared to a reference strain. The cell growth and viability were also significantly inhibited in the mgsAOE strain. Likewise in the speBOE or yaaOOE strains, the cell length in the mgsAOE strain was observed ranging from 2.5- to 3-fold longer than the reference strain. Additionally, the mreB gene expression increased similar to the speBOE or yaaOOE strains in similar to the mgsAOE strain.
Taken together, these results suggested that cellular methylglyoxal and polyamines are involved in the cell elongation as important factors reciprocally during the B. subtilis cell growth.