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Title Page

Contents

List of Abbreviations 12

English Abstract 14

Chapter 1. Introduction 17

1-1. General Background to Streptomyces 17

1-1-1. General biological and Genetic information on Streptomyces griseus and Streptomyces coelicolor. 18

1-1-2. Morphological differentiation in Streptomyces griseus and Streptomyces coelicolor 23

1-1-3. Life cycle of Streptomyces 23

1-1-4. Secondary metabolism in Streptomyces 24

1-2. SGT (Streptomyces griseustrypsin)(이미지참조) 28

1-3. Signal Transduction in bacteria 29

1-3-1. Eukaryotic-type Serine/Threonine protein kinases (STPKs) 30

1-3-1-1. STPKs in S. coelicolor 31

1-3-1-2. PASTA domains 31

1-3-2. Two-component system 32

1-4. Research Objectives 37

Chapter 2. Materials and Methods 38

2-1. Bacterial strains and Plasmids 38

2-1-1. E.coli strains 38

2-1-2. Streptomyces Strains 39

2-1-3. Plasmids 39

2-2. Growth Conditions and Storage of Bacterial Strains 40

2-2-1. E.coli strains 40

2-2-2. Streptomyces strains 40

2-2-3. Antibiotic concentrations for E.coli and streptomyces strains 40

2-3. Culture media, Buffers, Solutions, Reagents, and Enzymes 41

2-3-1. Solid media 41

2-3-2. Liquid Media 44

2-3-3. Buffers and solutions 46

2-4. Nucleic acid isolation 49

2-4-1. Isolation of plasmid DNA from E.coli 49

2-4-2. Isolation of genomic DNA from Streptomyces 49

2-4-3. Isolation of plasmid DNA from Streptomyces 50

2-4-4. Isolation of total RNA from Streptomyces 51

2-5. Genetic Manipulations 51

2-5-1. Preparation of competent E.coli cells 51

2-5-2. Transformation of E.coli competent cells 52

2-5-3. Transformation of Streptomyces protoplasts with plasmid DNA 52

2-5-3-1. Preparation of protoplasts 52

2-5-3-2. PEG-assisted transformation of Streptomyces protoplasts with plasmid DNA 53

2-5-3-3. Selection of antibiotic-resistant transformants by flooding 53

2-6. In Vitro Manipulations of DNA 53

2-6-1. Dephosphorylation (CIAP treatment) 53

2-6-2. Agarose gel electrophoresis of DNA and RNA 54

2-6-3. Isolation of DNA fragment from agarose 54

2-6-4. Ligation of DNA 54

2-6-5. cDNA synthesis 54

2-6-6. PCR 55

2-7. Gene disruption of pkaF in S. coelicolor 56

2-8. Assay 57

2-8-1. Sample preparation for proteaseassay 57

2-8-2. Determination of trypsin activity 58

2-8-3. Preparation of Cell Lysate from S. coelicolorA3(2) M130 58

2-8-4. Phosphorylation assay 58

Chapter 3. Characterization of the sgtR1 and sgtR2 genes and their role in regulating expression of the sprT gene encoding Streptomyces griseus trypsin (SGT) 60

3-1. Identification and characterization of the sgtR1 and sgtR2 genes 61

3-1-1. In silico analysis of the sgtR1 and sgtR2 genes. 61

3-1-2. Molecular analysis of the sgtR1 and sgtR2 genes 65

3-2. Overexpression of sprT in combination with sgtR1 and sgtR2 in S. lividans TK24 68

3-2-1. Construction of sprT expression vectors. 68

3-2-2. Determination of Trypsin activity in S.lividans strains 68

3-2-3. Transcriptional analysis of sprT, sgtR1, and sgtR2 in S.lividans Strains. 69

3-3. Overexpression of SGT with sgtR1 and sgtR2 in S. griseus 75

3-3-1. Determination of Trypsin activity in S.griseus 76

3-3-2. Transcriptional analysis of sprT, sgtR1, and sgtR2 in S.griseus strains 77

3-4. Summary 81

Chapter 4. Charcterization of the autophosphorylating kinase, PkaF, in Streptomyces coelicolorA3(2) M130 82

4-1. Identification and characterization of pkaF gene 82

4-1-1. In silico analysis of STPK domain 83

4-1-2. In silico analysis of PASTA domain 84

4-1-3. Gene organization in the cloned fragments containing pkaF from S. coelicolor 84

4-2. Heterologous expression and purification of pkaF in E. coliBL21(DE3) 87

4-3. Phosphorylation of PkaF 94

4-3-1. Phosphorylation assay of PkaF 94

4-3-2. Mass spectrometry of phospho-PkaF 95

4-4. Effect of pkaF-overexpression and disruption on S. coelicolorA3(2) 100

4-5. Discussrion 107

4-6. Summary 111

Appendix : DNA and protein sequence of sprT, sgtR1, and sgtR2 in S.griseus 112

References 114

Korean Abstract 123

List of Tables

Table 1-1. General features of the S.griseus chromosome(Ohnishi, Ishikawa et al. 2008). 21

Table 1-2. General features of the S.coelicolor chromosome (Bentley, Chater et al. 2002). 22

Table 1-3. The S.coelicolor STPKs(Petrickova and Petricek, 2003) 36

Table 2-1. Primers used in this work 55

Table 4-1. Peptide fingerprinting of the PkaF using MALDI-TOF mass spectrometry of the in-gel tryptic digested of the protein 92

Table 4-2. Tryptic peptides containing a single phosphate, as determined MALDI-TOF mass. 99

List of Figures

Figure 1-1. Schematic representation of the S.griseus chromosome (Ohnishi, Ishikawa et al. 2008). 19

Figure 1-2. Circular representation of the S.coelicolorchromosome(Bentley, Chater et al. 2002). 20

Figure 1-3. Life cycle of Streptomycetes 26

Figure 1-4. Secondary metabolism in Streptomyces griseus(Horinouchi 2007) 27

Figure 1-5. AfsK/AfsR system in S.coelicolor 34

Figure 1-6. Two-component system in Prokaryotes. 35

Figure 3-1. Organization of the sprT, sgtR1, and sgtR2 genes. 63

Figure 3-2. Distribution of conserved domains in SgtR1 and SgtR2. 64

Figure 3-3. Construction vector for overexpression of SgtR1(ORF2) and SgtR2(ORF3). 66

Figure 3-4. Overexpression of SgtR1(ORF2) and SgtR2(ORF3). 67

Figure 3-5. Construction of sprT expression vectors containing various combinations of sgtR1 and sgtR2. 70

Figure 3-6. Cell growth of S.livadans Transformants 71

Figure 3-7. Trypsin activities of S. lividans transformed with various expression vectors. 72

Figure 3-8. Protein hydrolysate assay on R2YE agar plates 73

Figure 3-9. Transcriptional analysis of S. lividans transformed with various expression vectors. 74

Figure 3-10. Trypsin activities of S. griseus transformed with various expression vectors. 78

Figure 3-11. Cell growth of S.griseus transformants 79

Figure 3-12. Transcriptional analysis of S. griseus transformed with various expression vectors. 80

Figure 4-1. Sequence analysis of PkaF. 85

Figure 4-2. Sequence analysis of PkaF. 86

Figure 4-3. Map of recombinant plasmid vector for PkaF overexpression 88

Figure 4-4. Overexpression and purification of PkaF. 89

Figure 4-5. Mass spectrometry analysis of PkaF protein. 93

Figure 4-6. Phosphorylation assay of the purified PkaF. 96

Figure 4-7. Phosphorylation assay of the purified PkaF. 97

Figure 4-8. Phosphorylation assay of the purified PkaF. 98

Figure 4-9. Effect of pkaF-overexpression and disruption on S. coelicolorA3(2) 102

Figure 4-10. Generation of a pkaF-deficient mutant strain. 103

Figure 4-11. Analysis of chromosomal DNA by Southern blot hybridization. 104

Figure 4-12. Transcriptional analysis of pkaF. 105

Figure 4-13. Photographs of the surface of the colony. 106

참고문헌 (74건) : 자료제공( 네이버학술정보 )

참고문헌 목록에 대한 테이블로 번호, 참고문헌, 국회도서관 소장유무로 구성되어 있습니다.
번호 참고문헌 국회도서관 소장유무
1 The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis 네이버 미소장
2 Acetylation of pronase trypsin 네이버 미소장
3 The role of Two-component regulation systems in the physiology of the bacterial cell 네이버 미소장
4 Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). 네이버 미소장
5 Regulation of secondary metabolism in streptomycetes 네이버 미소장
6 Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. 네이버 미소장
7 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding 네이버 미소장
8 The SmtB/ArsR family of metalloregulatory transcriptional repressors: structural insights into prokaryotic metal resistance 네이버 미소장
9 A nickel-cobalt-sensing ArsR-SmtB family repressor. Contributions of cytosol and effector binding sites to metal selectivity. 네이버 미소장
10 Genetics of Differentiation in Streptomyces 네이버 미소장
11 Signalling early developmental events in two highly diverged Streptomyces species 네이버 미소장
12 (2001). "Overexpression of SGPA and SGT induces morphological changes in Streptomyces lividans." J Microbiol Biotechnol 11: 1077-1086. 미소장
13 Sampling the Antibiotic Resistome 네이버 미소장
14 Induction of microbial secondary metabolism. 네이버 미소장
15 An improved aminoglycoside resistance gene cassette for use in gram-negative bacteria and Streptomyces. 네이버 미소장
16 Improved detection of helix-turn-helix DNA-binding motifs in protein sequences. 네이버 미소장
17 RegB/RegA, a highly conserved redox-responding global two-component regulatory system. 네이버 미소장
18 The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth. 네이버 미소장
19 Phosphorylation of Isocitrate Dehydrogenase of Escherichia coli 네이버 미소장
20 Positive Control of a Global Antioxidant Defense Regulon Activated by Superoxide-Generating Agents in Escherichia coli 네이버 미소장
21 Studies on transformation of Escherichia coli with plasmids 네이버 미소장
22 Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. 네이버 미소장
23 How do antibiotic‐producing bacteria ensure their self‐resistance before antibiotic biosynthesis incapacitates them? 네이버 미소장
24 Conjugative sex plasmids of Streptomyces. 네이버 미소장
25 A microbial hormone, A-factor, as a master switch for morphological differentiation and secondary metabolism in Streptomyces griseus. 네이버 미소장
26 AfsR as an integrator of signals that are sensed by multiple serine/threonine kinases in Streptomyces coelicolor A3(2) 네이버 미소장
27 Mining and Polishing of the Treasure Trove in the Bacterial Genus Streptomyces 네이버 미소장
28 Sensing and responding to diverse extracellular signals? Analysis of the sensor kinases and response regulators of Streptomyces coelicolor A3(2). 네이버 미소장
29 A bacterial environmental sensor that functions as a protein kinase and stimulates transcriptional activation. 네이버 미소장
30 Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. 네이버 미소장
31 The Mycobacterium tuberculosis serine/threonine kinases PknA and PknB: substrate identification and regulation of cell shape. 네이버 미소장
32 Transcriptional control by A-factor of two trypsin genes in Streptomyces griseus. 네이버 미소장
33 Control by A-factor of a metalloendopeptidase gene involved in aerial mycelium formation in Streptomyces griseus. 네이버 미소장
34 Initiation of aerial mycelium formation in Streptomyces 네이버 미소장
35 Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene 네이버 미소장
36 Effect of a global regulatory gene, afsR2, from Streptomyces lividans on avermectin production in Streptomyces avermitilis 네이버 미소장
37 Characterization of the genes and proteins of a two-component system from the hyperthermophilic bacterium Thermotoga maritima. 네이버 미소장
38 Aha1 binds to the middle domain of Hsp90, contributes to client protein activation, and stimulates the ATPase activity of the molecular chaperone. 네이버 미소장
39 Phosphorylation of Bacterial Response Regulator Proteins by Low Molecular Weight Phospho-Donors 네이버 미소장
40 Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector 네이버 미소장
41 Signaling by the Arc Two-Component System Provides a Link Between the Redox State of the Quinone Pool and Gene Expression 네이버 미소장
42 Analysis of the protein-kinase activity of Escherichia coli cells 네이버 미소장
43 CD-Search: protein domain annotations on the fly. 네이버 미소장
44 Phosphorylation of the AfsR protein involved in secondary metabolism in Streptomyces species by a eukary otic-type protein kinase 네이버 미소장
45 Modulation of superoxide generation in in vivo lipopolysaccharide-primed rat alveolar macrophages by arachidonic acid and inhibitors of protein kinase C, phospholipase A2, protein serine-threonine phosphatase(s), protein tyrosine kinase(s) and phosphatase(s). 네이버 미소장
46 Aha, Another Regulator for Hsp90 Chaperones 네이버 미소장
47 Ultrastructural Studies on Sporulation in Wild-type and White Colony Mutants of Streptomyces coelicolor 네이버 미소장
48 Tyrosine protein phosphorylation is required for protein kinase C-mediated proliferation in T cells 네이버 미소장
49 Substrate activation and substrate inhibition of trypsin-like enzymes from three strains of Streptomyces species. 네이버 미소장
50 Studies on proteolytic enzymes (pronase) of Streptomyces griseus K-1. II. Separation of exo- and endopeptidases of pronase. 네이버 미소장
51 StoPK‐1, a serine/threonine protein kinase from the glycopeptide antibiotic producer Streptomyces toyocaensis NRRL 15009, affects oxidative stress response 네이버 미소장
52 A sepharose derivative coupled with a leupeptin-like peptide aldehyde, glycylglycyl-L-argininal, and its use as an affinity adsorbent for trypsin. 네이버 미소장
53 Genome sequence of the streptomycin-producing microorganism Streptomyces griseus IFO 13350. 네이버 미소장
54 AdpA, a Central Transcriptional Regulator in the A-Factor Regulatory Cascade That Leads to Morphological Development and Secondary Metabolism in Streptomyces griseus 네이버 미소장
55 Targeting Two-Component Signal Transduction: A Novel Drug Discovery System 네이버 미소장
56 Amino acid sequence of Streptomyces griseus trypsin. Cyanogen bromide fragments and complete sequence. 네이버 미소장
57 Enzymic and physicochemical properties of Streptomyces griseus trypsin. 네이버 미소장
58 Engineering the primary substrate specificity of Streptomyces griseus trypsin. 네이버 미소장
59 Activation of the ATPase Activity of Hsp90 by the Stress-Regulated Cochaperone Aha1 네이버 미소장
60 Eukaryotic-type protein kinases in Streptomyces coelicolor: variations on a common theme. 네이버 미소장
61 Changes in Protein Kinase C Isoenzyme Expression Associated with Apoptosis in U937 Myelomonocytic Cells 네이버 미소장
62 Critical evaluation of comparative model building of Streptomyces griseus trypsin. 네이버 미소장
63 Refined crystal structure of Streptomyces griseus trypsin at 1.7 Å resolution 네이버 미소장
64 Self-activation of serine/threonine kinase AfsK on autophosphorylation at threonine-168. 네이버 미소장
65 Three chymotrypsin genes are members of the AdpA regulon in the A-factor regulatory cascade in Streptomyces griseus. 네이버 미소장
66 Antigenicity of proteinases from Streptomyces griseus (pronase). 네이버 미소장
67 The trypsin-like enzyme from Streptomyces griseus (pronase). 네이버 미소장
68 Autophosphorylation of a bacterial serine/threonine kinase, AfsK, is inhibited by KbpA, an AfsK-binding protein. 네이버 미소장
69 Protein serine/threonine kinases in signal transduction for secondary metabolism and morphogenesis in Streptomyces 네이버 미소장
70 Evidence for protein kinase activities in the prokaryote Salmonella typhimurium. 네이버 미소장
71 Development and Organization of the Aerial Mycelium in Streptomyces coelicolor 네이버 미소장
72 Septation During Sporulation in Streptomyces coelicolor 네이버 미소장
73 The PASTA domain: a beta-lactam-binding domain. 네이버 미소장
74 Identification of a putative eukaryotic-like protein kinase family in the developmental bacterium Myxococcus xanthus. 네이버 미소장

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