Glyoxylate bypass governs bacterial respiration under oxidative stress
산화스트레스 조건아래 세균의 호흡 조절에 관여하는 Glyoxylate 경로에 대한 연구
- 주제(키워드) glyoxylate shunt , isocitrate lyase , oxidative stress , denitrification , Pseudomonas aeruginosa
- 발행기관 고려대학교 대학원
- 지도교수 박우준
- 발행년도 2016
- 학위수여년월 2016. 2
- 학위구분 석사
- 학과 대학원 환경생태공학과
- 세부전공 생물재료공학 전공
- 원문페이지 102 p
- 실제URI http://www.dcollection.net/handler/korea/000000065745
- 본문언어 영어
- 제출원본 000045867234
초록/요약
The glyoxylate shunt is an alternative to the TCA cycle that bypasses the carbon dioxide-producing steps and is essential for acetate and fatty acid metabolism. The glyoxylate shunt is upregulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, the isocitrate lyase (aceA) and malate synthase (glcB) genes are not in an operon, unlike in Escherichia coli; thus, two genes might exhibit different transcriptional responses to stress. Contrary to our expectations, deletion of aceA from P. aeruginosa improved cell growth under oxidative and antibiotic stress, which could be attributed to decreased NADH production. Transcriptome data suggested that aceA mutants underwent a metabolic shift to aerobic denitrification, supported by additional evidence: upregulation of denitrification-related genes, decreased oxygen consumption without ATP reduction, increased production of denitrification intermediates, NO and N2O, and increased cyanide resistance. The aceA mutants could also overproduce a thicker exopolysaccharide layer, the phenotype related with aerobic denitrification. A global bioinformatics analysis showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. Our data suggest that the presence of isocitrate lyase, an aerobic adaptation, appears to allow cells to bypass the TCA cycle, not only to preserve the carbon source, but also to tolerate oxidative stress.
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ABSTRACT i
ABSTRACT IN KOREAN iii
ACKOWNOWLEDGMENT v
TABLE OF CONTENTS vi
LIST OF TABLES viii
LIST OF FIGURES ix
1. INTRODUCTION 1
2. MATERIALS AND METHODS 13
2.1. Antibiotics, culture media, bacterial strains, and growth conditions 13
2.2. Gene expression analysis using qRT-PCR 13
2.3. Survival and sensitivity of P. aeruginosa 14
2.4. Microarray analysis 14
2.5. Measurement of oxygen consumption rate 15
2.6. Detection of NO 15
2.7. Detection of N2O concentration 16
2.8. Biofilm formation assay 16
2.9. EPS analysis 16
2.10. Confocal laser scanning microscopy (CLSM) 17
2.11. Determination of ATP concentrations 17
2.12. Measurement of the NADH/NAD+ ratio 18
2.13. Determination of cellular acid-soluble thiols 18
2.14. Bioinformatic analysis of the glyoxylate shunt 19
3. RESULTS 20
3.1. Physiological and phenotypic alterations in the absence of the glyoxylate bypass 20
3.2. Transcriptomic and biochemical analyses of aceA mutants 31
3.3. Enhanced biofilm formation and pigment production in aceA mutants 43
3.4. Intracellular NADH level with PQ treatment 50
3.5. Total thiol levels under ROS stress 54
3.6. Phylogenetic distribution of the glyoxylate bypass in bacteria 60
4. DISCUSSION 66
5. CONCLUDING REMARKS 72
6. REFERENCES 74
