14 research outputs found
패혈증 비브리오균 바이오필름의 대사체적 분석과 바이오필름 제어 타겟의 확인
학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2015. 2. 최상호.Biofilms are assemblages of microbial cells enclosed in self-produced extracellular polymeric matrix (EPM). Biofilm formation provides Vibrio vulnificus with resistances to antimicrobial agents and a variety of stresses in the environment. In order to gain insight into the cellular mechanisms in mature biofilms, the global metabolite profiling of V. vulnificus biofilms and planktonic cells was analyzed by using gas chromatography-time of flight mass spectrometry (GC-TOF-MS) and multivariate statistical analysis. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) showed significant differences in metabolic profiles between biofilms and planktonic cells. The levels of some amino acids, monosaccharides and nucleotides were elevated in biofilms compared to the planktonic counterparts. These metabolites appear to function as building blocks for structural components of extracellular matrix. Metabolites involved in the anaerobic energy production, such as glycolysis and pyruvate fermentation, were increased in biofilms. These suggest that V. vulnificus may obtain energy by using glycolysis and pyruvate fermentation in biofilms. The amounts of product of putrescine biosynthetic pathway in bioflms were also greater than in the planktonic cells. These results suggested that putrescine may have a positive effect on formation of V. vulnificus biofilms. Furthermore, the amounts and expression levels of the metabolites and genes, respectively, involved in N-acetylglucosamine (GlcNAc) utilization were highly increased in V. vulnificus biofilms. The combined results suggest that, when V. vulnificus forms mature biofilms and encounters conditions of low-nutrient, GlcNAc-specific phosphotransferase system (PTS) may transfer GlcNAc which is derived from extracellular environments. To find out whether GlcNAc-specific PTS affects biofilm forming ability of V. vulnificus, biofilm forming activities of the wild type and GlcNAc-specific phosphotransferase (ptsG) mutant were compared. Under the condition of low carbon source, biofilm forming activity was reduced in the ptsG mutant compared to the wild type and recovered in the complemented strain. Composition analysis of exopolysaccharide (EPS) using GC-TOF-MS revealed that GlcNAc is one of the constituent of EPS. In these aspects, when bacterial cells in V. vulnificus biofilms are faced with adverse conditions, such as insufficient nutrient, PtsG appears to transport GlcNAc, as a carbon source, which stems from extracellular matrix. Consequently, V. vulnificus in biofilms exhibits a distinct mode of growth characterized by matrix components syntheses, anaerobic energy production, polyamine metabolism and N-acetylglucosamine utilization.Abstract…………………………………………………………………………….Ⅰ
Contents…………………………………………………………………………….ⅢList of Figures……………………………………………………………………...Ⅴ
List of Tables………….……………………………………………………………Ⅵ
I. INTRODUCTION………………………………………………………………...1
II. MATERIALS AND METHODS………………………………………………...4
Culture conditions and biofilm formation……………………………..………….4
Metabolites extraction …….…………………………………………………….. 5
Derivatization of metabolites …………………………….………………………5
GC-TOF-MS analysis..........…………...…….………………..………………….6
Data processing.....………………….…………….…………………………...…6
Statistical analysis ………………………………………………………………..7
Generation of the V. vulnificus ptsG mutant …………….………………………7
Genetic complementation of ptsG null mutant ….……………………………….8
Extraction and analysis of EPS ….……………………………..………………...9
III. RESULT……………………………………………………………………….12
Biofilm formation in the glycerol containing minimal media ………………….12
Schematic work flow of metabolomics study …………..………………………14
Multivariate statistical analysis of biofilms and plnktonic cells ………………..18
Identification of metabolites from standards and libraries …….………………..20
Carbohydrate metabolism pathway analysis ……………………...…………….27
Putrescine biosynthetic pathway analysis ………………………….…….……..29
N-acetylglucosamine, an important carbon source in V. vulnificus biofilms...….35
Composition analysis of wild type V. vulnificus EPS …………………………..35
Construction and confirmation of V.vulnificus ptsG mutant ……………..…….37
Bofilm-forming activity of wild type and ptsG mutant under various culture
conditions ...........…………………………….………………………………….40
IV. DISCUSSION……………………………………………………………….…42
V. REFERENCES………………………………………………………………….46
VI. 국문초록………………………………………………………………………53Maste