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Regulation and Function of Redox Sensor SoxR in Streptomyces coelicolor : Streptomyces coelicolor에서 산화환원 센서인 SoxR의 기능과 조절

DC Field Value Language
dc.contributor.advisorJung-Hye Roe-
dc.contributor.author싱아툴쿠마르-
dc.date.accessioned2017-07-14T00:47:47Z-
dc.date.available2017-07-14T00:47:47Z-
dc.date.issued2014-08-
dc.identifier.other000000020695-
dc.identifier.urihttps://hdl.handle.net/10371/121390-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2014. 8. Jung-Hye Roe.-
dc.description.abstractRedox-sensitive transcription factor SoxR in enteric bacteria regulates cellular response toward superoxide and nitric oxide via inducing the expression of a downstream regulator SoxS, that activates more than 100 genes. In other bacterial groups, however, SoxR directly induces its multiple target genes in response to redox-active compounds, as initially demonstrated for psuedomonads. The antibiotic-producing soil bacterium Streptomyces coelicolor contains a gene for SoxR homologue (SCO1697) whose DNA-recognition helix is identical to that of Escherichia coli SoxR. Using E. coli SoxR binding sequence, five candidate genes of SoxR reglulon were predicted. It was demonstrated that SoxR binds to their promoter regions and activates their expression concurrently with the production of blue polyketide antibiotic actinorhodin (a benzoisochromanequinone). These genes encode probable NADPH-dependent flavin reductase (SCO2478), NADPH-dependent quinone reductase (SCO4266), ABC-transporter (SCO7008), monooxygenase (SCO1909), and a hypothetical protein (SCO1178). Addition of actinorhodin to exponentially growing cells activated the expression of SoxR target genes in a SoxR-dependent manner.
SoxR from E. coli and related enterobacteria is activated by a broad range of redox-active compounds through oxidation or nitrosylation of its [2Fe-2S] cluster. In contrast, non-enteric SoxRs appear to get activated by a narrower range of redox-active compounds that include endogenously produced metabolites. The responsiveness of SoxRs from Streptomyces coelicolor (ScSoxR), Pseudomonas aeruginosa (PaSoxR) and E. coli (EcSoxR), all expressed in S. coelicolor, were compared toward natural or synthetic redox-active
compounds. EcSoxR responded to all compounds examined, whereas ScSoxR was insensitive to oxidants such as paraquat (Eh -440 mV) and menadione sodium bisulfite (Eh -45 mV) and to nitric oxide (NO) generators. PaSoxR was insensitive only to some NO generators. Whole cell EPR analysis of SoxRs expressed in E. coli revealed that the [2Fe-2S]1+ of ScSoxR was not oxidizable by paraquat, differing from EcSoxR and PaSoxR. The mid-point redox potential of purified ScSoxR was determined to be -185 ± 10 mV, higher by ~100 mV than those of EcSoxR and PaSoxR, coinciding with its insensitivity to paraquat. The overall sensitivity profile indicates that both redox potential and kinetic reactivity determine the differential responses of SoxRs toward various oxidants. Residues within the [2Fe-2S] binding site, which are specific to ScSoxR, were mutated and were evaluated for their effects on the sensitivity profile.


Key Words		-
dc.description.abstractStreptomyces coelicolor, SoxR, Fe-S, redox-active compounds, EPR, redox potential, oxidative stress, superoxide-
dc.description.tableofcontentsAbstract i
Contents iii
List of figures vii
List of tables x
Abbreviations xi

Chapter I. Introduction 1
I.1. Biology of Streptomyces coelicolor 2
I.2. Oxidative stress responses 3
I. 3. Reactive oxygen species. 5
I.3.1. Superoxide radical (O2-) 7
I.3.2. Hydrogen peroxide (H2O2) 7
I.3.3. Hydroxyl radical (HO•) 8
I.3.4. Singlet oxygen (1g O2) 8
I.4. Redox-active compounds 9
I. 5. Mechanisms of oxidative cell damage. 10
I.5.1.Biological defense systems to oxidative stress 11
I.5.2. SoxR and the SoxRS response to superoxide stress in E. coli 13
I.5.3. Salmonella typhimurium SoxR 21
I.5.4. Pseudomonas SoxR 21
I.5.5. Xanthomonas campestris SoxR 25
I.5.6. S. coelicolor SoxR 25
I. 6. Aims of this study. 29

Chapter II. Materials and Methods 30
II.1. Bacterial strains and culture conditions 31
II.1.1. Streptomyces coelicolor 31
II.1.2. Escherichia coli 31
II.1.3. Pseudomonas aeruginosa 32
II.2. Chemical treatments 34
II.3. DNA manipulation 34
II.3.1. DNA isolation and purification 34
II.3.2. General recombinant DNA techniques 34
II.3.3. DNA sequencing 35
II.3.4. Polymerase chain reaction (PCR) 35
II.4.PCR-targeted disruption of genes in S.coelicolor 35
II.4.1. Construction of DsoxR mutant 35
II.4.2. Construction of DsoxR strains expressing ScSoxR, EcSoxR, or PaSoxR 36
II.4.3. Construction of truncated and swapped ScSoxR 37
II.4.4. Electrophoretic mobility shift assay (EMSA) for SoxR-DNA binding. 38
II.4.5. S1 nuclease mapping analysis. 39
II.5. Protein purification 39
II.5.1. Overproduction and purification of S. coelicolor SoxR protein from E.coli 39
II.5.2. Enzyme activity assay 40
II.5.2.1. β-Galactosidase (LacZ) assay 40
II.6. Biochemical assays 41
II.6.1. UV-visible absorption spectrometry 41
II.6.2. Electron paramagnetic resonance (EPR) spectroscopy of SoxR 41
II.6.3. Redox titration of SoxR 42
II.7. Methods for bioinformatic analyses 42
II.7.1. Genome databases 42
II.7.2. Analysis of sequence and structure 42
II.8. Site-specific mutagenesis of SoxR 43

Chapter III. Results 46

III.1. Characterization of S.coelicolor [2Fe-2S] SoxR protein 47
III.1.1. In vitro properties of SoxR wild type and cysteine to serine substitution mutant proteins. 47
III.1.2. Dimerization of SoxR 47
III.1.3. UV-VIS absorption spectrum and EPR characteristic of [2Fe-2S] containing proteins 48
III.1.4. The [2Fe-2S] clusters in SoxR are essential for transcriptional activity 49

III.2. Comparative study of SoxR activation by redox-active compounds 57
III.2.1. Induction of ScSoxR by both natural and xenobiotic redox active compounds in S. coelicolor 57
III.2.2. SoxR protects cells from the growth-inhibiting effects of SoxR-inducing chemicals 63
III.2.3. Differential sensitivity profile of SoxRs toward RACs in S. coelicolor, E. coli, and P. aeruginosa 63
III.2.4.Activation profile of three SoxR species expressed in S.coelicolor or in E. coli by various RACs64
III.2.5. Time course of the activation of EcSoxR and PaSoxR by paraquat 73
III.2.6. In vivo redox status of [2Fe-2S] cluster of SoxRs following oxidant treatment 76
III.2.7. Measurement of Redox Potential of ScSoxR 77
III.3. Mutational analysis of Streptomyces coelicolor SoxR to define the regions required for redox active molecules sensing and transcriptional activation 84
III.3.1. Mutations in specific residues of S. coelicolor SoxR alters its specificity for redox-active molecules 84
III.3.2. In vivo redox status of [2Fe-2S] cluster of S. coelicolor SoxR, ScSoxR-L126R and SWAP-1 without treatment 95
III.3.3. Mutations in specific residues of E.coli SoxR alters its specificity toward paraquat (PQ) 95
III.3.4. Mutations in M. smegmatis SoxR around Fe-S cluster specific residue 96
III.3.5. Slower differentiation and decreased production of antibiotics in ∆soxR mutants 97

Chapter IV. Discussion 105
References 112
Acknowledgements 126		-
dc.formatapplication/pdf-
dc.format.extent4813402 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectMolecular Microbiology-
dc.subjectSoxR-
dc.subjectFe-S-
dc.subjectredox-active compounds-
dc.subjectEPR-
dc.subjectredox potential-
dc.subjectoxidative stress-
dc.subjectsuperoxide-
dc.subject.ddc570-
dc.titleRegulation and Function of Redox Sensor SoxR in Streptomyces coelicolor-
dc.title.alternativeStreptomyces coelicolor에서 산화환원 센서인 SoxR의 기능과 조절-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pagesxi, 140-
dc.contributor.affiliation자연과학대학 생명과학부-
dc.date.awarded2014-08-
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