표제지
Abstract
목차
서론 18
1. Corynebacterium glutamicum의 분류 및 특징 19
2. C. glutamicum의 stress responses 기작 21
3. Oxidative stress 대응 기작 네트워크 28
4. Cell envelope에서의 disulfide bond formation system 35
5. Coyrnebacterium glutamicum의 mycolic acid 39
재료 및 방법 44
1. 균주 및 배양 조건 45
2. 시약 45
3. 사용된 oligonucleotides 45
4. 연구에 사용된 plasmid 제작 59
4.1. pK19mobsacB cloning 59
4.2. pSL360 cloning 61
5. Electroporation 62
6. C. glutamicum RNA preparation and qRT-PCR 64
6.1. RNA sample preparation 및 cDNA conversion 64
6.2. Quantitative real time PCR유전자 64
7. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) 65
7.1. Sample preparation 65
7.2. Rehydration과 IEF 65
7.3. 2D-running과 staining 66
8. 세포 내 NADPH/NADP+ 및 NADH/NAD+ ratio 측정[이미지참조] 66
9. ChIP (chromatin immunoprecipitation) sequencing 67
9.1. Cross-linking and sample preparation 67
9.2. Immunoprecipitation 67
10. 외부 스트레스에 대한 physiological analysis 68
10.1. Kirby-Bauer test 68
10.2. Spot titer assay 68
10.3. Cell viability test 68
11. 단백질 정제에 사용된 plasmid들의 제작 69
11.1. pET28a-osnR (pSL581) 69
11.2. pMAL-c2-cdbC (pSL612) 69
12. 재조합 단백질의 정제 70
12.1. His6-OsnR protein[이미지참조] 70
12.2. MBP-CdbC and MBP-CdbCC98A protein[이미지참조] 70
13. EMSA (Electrophoretic Mobility Shift Assay) 71
14. Lac operon fusion system에 사용된 plasmid제작 71
15. β-galactosidase 활성의 측정 72
16. Site-directed mutagenesis 73
17. Protein disulfide isomerase assay 75
17.1. Insulin reduction assay 75
17.2. Scrambled RNaseA assay 75
18. Microscopy 76
18.1. Scanning electron microscopy (SEM) 76
18.2. Confocal microscopy 76
19. Classical MATH assay 77
20. Thin layer chromatography (TLC) 77
결과 78
I. OsnR is an auto-regulatory negative transcription factor controlling redox-dependent stress responses in Corynebacterium glutamicum 79
1. osnR (cg3230) 유전자 분리 및 분석 79
2. C. glutamicum osnR 결손 균주의 제작 (HL1638) 80
3. osnR 과발현 균주 제작 (HL1643) 83
4. osnR 유전자의 결손 및 과발현이 성장에 미치는 영향 86
5. osnR 결손 및 과발현 균주의 stress-causing agents sensitivity test 88
6. osnR 과발현의 영향을 받는 유전자 확인 92
7. osnR 돌연변이 균주에서 NADPH/NADP+ 및 NADH/NAD+ ratio측정[이미지참조] 96
8. C. glutamicum에서 osnR 유전자의 mRNA 발현량 변화 측정 98
9. osnR 과발현이 mycothiol metabolism에 미치는 영향 102
10. ChIP-seq.을 통한 osnR의 target 유전자 검출 106
11. His6-OsnR 재조합 단백질 정제[이미지참조] 111
12. OsnR 단백질을 이용한 EMSA 115
13. 산화적 환경에 따른 His6-OsnR 단백질-DNA 결합 변화[이미지참조] 121
14. osnR의 조절을 받는 상위 조절 유전자 규명 123
15. EMSA를 통한 OsnR단백질과 sigH promoter의 결합 확인 129
16. In vivo상의 결합 확인을 위한 lac operon fusion system 131
17. osnR 조절 유전자 확인을 위한 β-galactosidase 활성 측정 134
II. Functional caracterization of CdbC, a novel periplasmic protein disulfide-isomerase in Corynebacterium glutamicum. 138
1. CdbC (cg0026p) 단백질 특성 분석 138
2. cg0026 유전자와 OsnR의 발현 조절 관계 분석 141
3. MBP-CdbC 재조합 단백질 정제 143
4. In vitro에서 CdbC 단백질의 protein disulfide isomerase 기능 확인 147
5. C. glutamicum cdbC 결손 균주 제작 (HL1723) 150
6. cdbC 과발현 균주 제작 (HL1726) 153
7. Site-directed mutagenesis을 통한 cdbC C98A 과발현 균주 제작 (HL1749) 154
8. cdbC 결손 및 과발현이 성장에 미치는 영향 157
9. 산화적 스트레스 대응에서의 cdbC의 역할 확인 160
10. 다양한 스트레스에 대한 cdbC 변이균의 민감성 확인 161
11. cdbC가 cell wall metabolism에 미치는 영향 164
12. cdbC 변이균의 mycomembrane분석 167
13. cdbC 변이균의 mycolic acid 변화가 세포 형태에 미치는 영향 170
14. CdbC와 mycoloyltransferase의 연계성 예상 176
고찰 178
I. OsnR is an auto-regulatory negative transcription factor controlling redox-dependent stress responses in Corynebacterium glutamicum 179
II. Functional characterization of CdbC, a novel periplasmic protein disulfide-isomerase in Corynebacterium glutamicum 186
참고문헌 192
Table 1. Corynebacterium glutamicum sigma factor and its corresponding functions. 27
Table 2. WhiB-like proteins in C. glutamicum. 32
Table 3. Composition of medium 46
Table 4. List of bacterial strains 47
Table 5. List of plasmids 49
Table 6. List of oligonucleotides 51
Table 7. Ratio of NADH/NAD+ and NADPH/NADP+ in C. glutamicum strains.[이미지참조] 97
Figure 1. Scanning Electron Microscopy of Corynebacterium glutamicum. 20
Figure 2. Regulatory modules activated under acidic conditions in C. glutamicum. 25
Figure 3. A model of the regulatory network controlling expression of the HSP genes in response to heat shock. 26
Figure 4. The transcriptional regulatory network consisting of σH and transcription factors in the σH regulon.[이미지참조] 33
Figure 5. The TRX/MSH system. 34
Figure 6. Disulfide bond formation in the periplasm. 38
Figure 7. Schematic representation of the cell envelope of Corynebacterium glutamicum. 41
Figure 8. Lipid metabolism and its proposed regulatory mechanism in C. glutamicum. 42
Figure 9. Putative model for mycoloyltransferases functions and localizations in C. glutamicum. 43
Figure 10. Genetic maps of osnR (A) and cdbC (B) genes in C. glutamicum and the complementary sequences of primers. 60
Figure 11. Genetic map of myc tag primer (A) and restriction recognition site (B). 63
Figure 12. Schematic diagram of PCR-based site-directed mutagenesis. 74
Figure 13. Map of pSL578 and verification in agarose gel 81
Figure 14. Illustration of gene deletion by double crossover event. 82
Figure 15. Construction of the osnR-overexpressing strain. 84
Figure 16. The mRNA expression level of osnR gene using qRT-PCR. 85
Figure 17. Growth of characteristics of C. glutamicum strains. 87
Figure 18. Sensitivity of C. glutamicum strain to oxidants and antibiotics. 90
Figure 19. Sensitivity of C. glutamicum strains to lysozyme, SDS and temperature. 91
Figure 20. Expression of genes involved in oxidative stress responses. 94
Figure 21. Identification of proteins under control of the osnR gene by 2D-PAGE. 95
Figure 22. Quantification of osnR gene in C. glutamicum by qRT-PCR. 100
Figure 23. The transcription of osnR after challenge with stress-causing agents. 101
Figure 24. The mRNA levels of mycothiol biosynthetic genes in C. glutamicum cells. 104
Figure 25. The sensitivities of the C. glutamicum cells to alkylating agents and antibiotics. 105
Figure 26. Verification of myc-osnR overexpression strain. 108
Figure 27. Confirmation of myc-osnR overexpression and antibody binding test. 109
Figure 28. Target sites of the OsnR protein, as identified through the ChIP-seq analysis, and transcription levels of the identified genes in C. glutamicum cells. 110
Figure 29. Construction of the pET28a-osnR plasmid. 112
Figure 30. Overexpression and purification of His6-OsnR fusion protein. 113
Figure 31. Purification of His6-OsnR fusion protein. 114
Figure 32. Binding of the purified OsnR protein to the promoter region of the osnR gene. 118
Figure 33. DNA binding of the purified His6-OsnR protein to the promoter regions of the putative target genes identified by ChIP-seq.[이미지참조] 119
Figure 34. DNA binding of the purified His6-OsnR protein to the promoter regions of the putative target genes identified by qRT-PCR.[이미지참조] 120
Figure 35. Effects of diamide and DTT on the DNA-binding activity of OsnR. 122
Figure 36. Quantification of mRNA levels of whiB-like genes and genes effected by whcA in C. glutamicum cells. 126
Figure 37. The transcription levels of σ-factor-encoding genes in C. glutamicum cells. 127
Figure 38. The transcription levels of σ-factor-encoding genes after challenge with H₂O₂. 128
Figure 39. Binding of the purified OsnR protein to the promoter region of the sigH gene. 130
Figure 40. Vector map of pSL594, pSL595 and pSL596. 132
Figure 41. Vector map of pSL592 and verification on agarose gel 133
Figure 42. Effects of diamide and DTT on the DNA-binding activity of OsnR. 136
Figure 43. In vivo assays showing the binding of the OsnR protein to the promoter region of the sigH gene. 137
Figure 44. Analysis of CdbC-encoded cg0026 protein sequence. 139
Figure 45. Multiple sequence alignment of cg0026 protein with other DsbA family proteins. 140
Figure 46. Effect of the reductant on the expression of osnR (A) and cg0026 gene (B). 142
Figure 47. Construction of the pMAL-c2-cdbC and pMAL-c2-cdbCC98A.[이미지참조] 144
Figure 48. Analysis of protein overexpression with or without signal sequence of CdbC. 145
Figure 49. Purification of MBP-CdbC and MBP-CdbCC98A fusion proteins.[이미지참조] 146
Figure 50. Disulfide bond isomerase activity of CdbC compared with CdbCC98A.[이미지참조] 149
Figure 51. Map of pSL602 and verification in agarose gel 151
Figure 52. Illustration of gene deletion by double crossover event. 152
Figure 53. Verification of cg0026 (cdbC) overexpression strain. 155
Figure 54. Construction of the P180-cdbCC98A strain.[이미지참조] 156
Figure 55. Growth of characteristics of C. glutamicum strains. 158
Figure 56. Growth characteristics of C. glutamicum strains added with reducing agent. 159
Figure 57. Sensitivity of C. glutamicum to oxidant and expression of genes involved in oxidative stress responses. 162
Figure 58. Sensitivity of the C. glutamicum strains to various stresses. 163
Figure 59. Measurement of cell surface hydrophobicity. 165
Figure 60. The transcription of fatty acid and mycolic acid biosynthesis genes as determined by qRT-PCR. 166
Figure 61. Analysis of cell envelope mycolic acid of C. glutamicum and cdbC mutant strains. 168
Figure 62. The transcription of mycoloyltransferase genes as determined by qRT-PCR 169
Figure 63. Scanning electron microscope images of C. glutamicum. 172
Figure 64. Histograms showing the distribution of cell lengths of C. glutamicum. 173
Figure 65. Confocal laser scanning microscopy of C. glutamicum strains. 174
Figure 66. The transcription of genes involved in cell division as determined by qRT-PCR. 175
Figure 67. Overall structure of MytA and MytC. 177
Figure 68. Protein structure prediction by the SWISS-MODEL. 184
Figure 69. Regulatory model of OsnR on the expression of oxidative stress response genes. 185
Figure 70. CdbC protein regulation model of the disulfide-isomerization pathway in cell envelope. 191