Interactions of TCA cycle enzymes and of the CcpA-HPrSer46P complex with various cre-elements in Bacillus subtilis: = Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis
Saved in:
| Main Author: | |
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2013
|
| Subjects: | |
| Online Access: | Volltext Volltext http://d-nb.info/1035000814/34 Inhaltsverzeichnis |
| Physical Description: | IV, 107 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Interactions of TCA cycle enzymes and of the CcpA-HPrSer46P complex with various cre-elements in Bacillus subtilis |b = Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis |c von Maike Bartholomae |
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Record in the Search Index
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| adam_text | IMAGE 1
TABLE OF CONTENTS
1 ZUSAMMENFASSUNG -1-
2 SUMMARY -2-
3 INTRODUCTION -3-
3.1 THE GRAM-POSITIVE MODEL ORGANISM BACILLUS SUBTILIS - 3 -
3.2 METABOION FORMATION IN B. SUBTILIS - 3 -
3.2.1 TCA CYDE AS CENTRAL PART OF METABOLISM AND ANABOLISM - 4 -
3.2.2 REGULATION OF MDH, ICD AND EFT? - 5 -
3.2.3 THE NADP* DEPENDENT ISOCITRATE DEHYDROGENASE LED - 7 -
3.2.4 THE MALATE DEHYDROGENASE MDH - 8 -
3.2.5 THE CITRATE SYNTHASE CITZ -9-
3.3 REGULATION OF CARBON METABOLISM OF B. SUBTILIS -11 -
3.3.1 THE PHOSPHOENOLPYRUVATE: SUGAR PHOSPHOTRANSFERASE SYSTEM (PEP:PT3)
-11 - 3.3.2 THE CATABOLITE CONTROL PROTEIN A AND CCR IN 8. SUBTILIS -12
-
3.3.3 THE CATABOLITE RESPONSIVE ELEMENT ERE -15 -
3.3.4 THE REGULATION OF ACKA, GNTR. XYLA AND XYNP -16 -
3.4 SCOPE OF THE THESIS -W-
4 MATERIAL AND METHODS -18-
4.1 MATERIAL -18 -
4.1.1 CHEMICALS -18-
4.1.2 AUXILIARY MATERIAL -20-
4.1.3 INSTRUMENTS -21-
4.1.4 REAGENT MIXTURES -22-
4.1.5 PROTEINS, ENZYMES AND STANDARDS - 22 -
4.1.6 OLIGONUCLEOTIDES - 23 -
4.2 BACTERIAL STRAINS - 24 -
4.3 PLASMIDS - 24 -
4.4 BUFFER AND MEDIA - 25 -
4.4.1 GENERAL BUFFERS AND SOLUTIONS_ - 25 -
4.4.2 BUFFERS AND SOLUTIONS FOR PROTEIN PURIFICATION - 25 -
4.4.3 ONA AND PROTEIN GEL ELECTROPHORESIS - 26 -
4.4.3.1 DNA GEL ELECTROPHORESIS - 26 -
4.4.3.2 PROTEIN GEL ELECTROPHORESIS - 26 -
4.4.4 MEDIA AND SUPPLEMENTS - 28 -
J
HTTP://D-NB.INFO/1038603099
IMAGE 2
TABLE OF CONTENTS
4.5 METHODS - 29 -
4.5.1 GENERAL METHODS -29-
4.5.2 GROWTH OF BACTERIA -29-
4.5.3 TRANSFORMATION OF E. COLI - 29 -
4.5.3.1 PREPARATION OF CACI 2 COMPETENT E. COLI CELLS - 29 -
4.5.3.2 TRANSFORMATION OF E. COLI - 30 -
4.5.3.3 LONG TIME STORAGE OF BACTERIAL STRAINS - 30 -
4.5.4 METHODS FOR PURIFICATION AND MODIFICATION OF NUCLEIC ACIDS - 30 -
4.5.4.1 HYBRIDIZATION OF ERE DNA - 30 -
4.5.4.2 ISOLATION OF CHROMOSOMAL DNA FROM 8. SUBTILIS - 31 -
4.5.4.3 ISOLATION OF PLASMID DNA FROM E. COLI - 31 -
4.5.4.4 POLYMERASE CHAIN REACTION (PCR) - 32 -
4.5.4.5 RESTRICTION OF DNA - 32 -
4.5.4.6 DEPHOSPHORYLATION OF ONA - 33 -
4.5.4.7 LIGATION OF DNA FRAGMENTS - 33 -
4.5.4.8 ELUTION OF ONA FROM AGAROSE GELS - 33 -
4.5.4.9 SEQUENCING OF DNA - 33 -
4.5.5 METHODS FOR PURIFICATION AND ANALYSIS OF PROTEINS - 34 -
4.5.5.1 DENATURATING PROTEIN GEL ELECTROPHORESIS (PAGE) - 34 -
4.5.5.2 NATIVE PAGE - 34 -
4.5.5.3 CELL CULTIVATION FOR PROTEIN OVEREXPRESSION - 34 -
4.5.5.4 SONICATION -34-
4.5.5.5 OVEREXPRESSION TEST - 35 -
4.5.5.6 PURIFICATION OF CCPA(HIS) B MDH(HIS)B LCD(HIS)B LCDS104P(HIS)6
AND CITZ(HIS)6 FROM BACILLUS
SUBTILIS - 35 -
4.5.5.7 PREPARATION OF E. COLI DH5A P4813/ HPR KINASE EXTRACT - 36 -
4.5.5.8 PURIFICATION OF HPRSER46P - 36 -
4.5.5.9 DIALYSIS OF PROTEIN SOLUTIONS - 37 -
4.5.5.10 SURFACE PLASMON RESONANCE (SPR) ANALYSIS - 37 -
4.5.5.11 LED ACTIVITY ASSAY -39-
4.5.5.12 MDH ACTIVITY ASSAY - 40 -
4.5.5.13 CITZ ACTIVITY ASSAY -40-
4.5.6 COMPUTER PROGRAMS, DATABASES AND ONLINE SERVICES -41 -
4.5.6.1 COMPUTER PROGRAMS - 41 -
4.5.6.2 DATABASES AND ONLINE SERVICES -41 -
II
IMAGE 3
TABLE OF CONTENTS
5 RESULTS -43 -
5.1 METABOLITE DEPENDENT INTERACTION OF ISOCITRATE DEHYDROGENASE WITH
MALATE
DEHYDROGENASE OF BACILLUS SUBTILIS - 43 -
5-1.1 PURIFICATION OF MDH(HIS) B LCD(HIS)6AND LCDS104P(HIS)S -43 -
5.1.2 INTERACTION OF MDH(HIS) T AND LCD(HIS)6 - 44 -
5.1.3 SPR ANALYSIS OF THE INTERACTION OF LCD(HIS) S AND MDH(HIS)6 IN THE
PRESENCE OF COFACTORS AND
SUBSTRATES - 45 -
5.1.3.1 SPR ANALYSIS OF LCD(HIS) E BINDING TO MDH(HIS)S IN THE PRESENCE
OF ISOCITRATE, A-KETOGLUTARATE,
MALATE AND OXALOACETATE - 45 -
5.1.3.2 INFLUENCE OF THE ENZYMATIC ACTIVITY OF LCD(HIS) S ON THE BINDING
TO MDH(HIS)6 -47 -
5.2 INTERACTION OF MDH(HLS) E AND ICDS104P(HIS)( - 48 -
5.2.1 ACTIVITY OF LCD(HIS) 6 AND LCDS104P(HIS)S -48 -
5.2.2 SPR ANALYSIS OF LCDS104P(HIS) E BINDING TO MDH(HIS)S -49 -
5.2.3 SPR ANALYSIS OF LCDS104P(HIS) S BINDING TO MDH(HIS)5 IN THE
PRESENCE OF ISOCITRATE, A-KETOGLUTARATE,
MALATE AND OXALOACETATE - 50 -
5.2.4 QUANTIFICATION OF THE INTERACTION BETWEEN LCDS104P(HIS) 6 AND
MDH(HIS)6 STIMULATED BY ISOCITRATE,
NADP* AND MGCI, - 52 -
5.3 MUTUAL INFLUENCE OF LED AND MDH ON THEIR ENZYMATIC ACTIVITY - 53 -
5.3.1 INFLUENCE OF LED AND LCDS104P ON MDH ACTIVITY - 54 -
5.3.2 INFLUENCE OF MDH ON THE ENZYMATIC ACTIVITY OF LED AND LCDS104P -
56 -
5.4 INTERACTION ANALYSIS OF MALATE DEHYDROGENASE AND CITRATE SYNTHASE -
58 -
5.4.1 PURIFICATION OF CITZ(HIS) 6 - 58 -
5.4.2 SPR ANALYSIS OF CITZ(HIS) 6 BINDING TO MDH(HIS)6 - 59 -
5.4.3 SPR-ANATYSIS OF CITZ(HIS) S AND MDH(HIS)6 INTERACTION IN THE
PRESENCE OF MALATE AND OXALOACETATE
-59-
5.5 INTERACTION OF VARIOUS CRE-ELEMENTS WITH THE CCPA-HPR5ER46P COMPLEX
- 61 - 5.5.1 PURIFICATION OF CCPA(HIS) T AND HPRSER46P -61-
5.5.1.1 PURIFICATION OF CCPA(HIS) S - 61 -
5.5.1.2 PURIFICATION OF HPRSER46P - 62 -
5.5.2 QUALITATIVE AND QUANTITATIVE ANALYSIS OF CCPA COEFFECTOR COMPLEXES
INTERACTING WITH VARIOUS CREELEMENTS - 63 -
5.5.2.1 SELECTION AND PREPARATION OF THE ANALYZED CRE-ELEMENTS - 63 -
5.5.2.2 QUALITATIVE ANALYSIS OF CCPA-HPRSER46P BINDING TO XYLA-CRE - 64
-
5.5.2.3 QUANTIFICATION OF THE CCPA HPRSER46P XYM-CRE INTERACTION -65 -
5.5.2.4 QUANTIFICATION OF CCPA HPRSER46P COMPLEXES INTERACTING WITH
XYNP-CRE, ACKA-CREL, GNTR CREDON AND SYN-CRE - 66 -
III
IMAGE 4
TABLE OF CONTENTS
6 DISCUSSION -69-
6.1 INDICATIONS FOR THE FORMATION OF A TCA CYCLE METABOLON - 69 -
6.2 STRUCTURE AND KINETICS OF THE CCPA-HPRSER46P COMPLEX INTERACTING
WITH VARIOUS CRE ELEMENTS - 75 -
6.3 OUTLOOK - 79 -
7 BIBLIOGRAPHY -80-
8 ABBREVIATION INDEX -102-
IV
|
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| discipline | Biologie |
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| spelling | Bartholomae, Maike Verfasser (DE-588)1036793648 aut Interactions of TCA cycle enzymes and of the CcpA-HPrSer46P complex with various cre-elements in Bacillus subtilis = Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis von Maike Bartholomae Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis 2013 IV, 107 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Erlangen-Nürnberg, Univ., Diss., 2013 Heubacillus (DE-588)4242465-3 gnd rswk-swf Genregulation (DE-588)4122166-7 gnd rswk-swf Citronensäurezyklus (DE-588)4148058-2 gnd rswk-swf Enzymaktivität (DE-588)4152470-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Heubacillus (DE-588)4242465-3 s Genregulation (DE-588)4122166-7 s Citronensäurezyklus (DE-588)4148058-2 s Enzymaktivität (DE-588)4152470-6 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:29-opus-46498 https://open.fau.de/handle/openfau/3367 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-46498 Resolving-System kostenfrei Volltext http://d-nb.info/1035000814/34 Langzeitarchivierung Nationalbibliothek DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026106421&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Bartholomae, Maike Interactions of TCA cycle enzymes and of the CcpA-HPrSer46P complex with various cre-elements in Bacillus subtilis = Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis Heubacillus (DE-588)4242465-3 gnd Genregulation (DE-588)4122166-7 gnd Citronensäurezyklus (DE-588)4148058-2 gnd Enzymaktivität (DE-588)4152470-6 gnd |
| subject_GND | (DE-588)4242465-3 (DE-588)4122166-7 (DE-588)4148058-2 (DE-588)4152470-6 (DE-588)4113937-9 |
| title | Interactions of TCA cycle enzymes and of the CcpA-HPrSer46P complex with various cre-elements in Bacillus subtilis = Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis |
| title_alt | Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis |
| title_auth | Interactions of TCA cycle enzymes and of the CcpA-HPrSer46P complex with various cre-elements in Bacillus subtilis = Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis |
| title_exact_search | Interactions of TCA cycle enzymes and of the CcpA-HPrSer46P complex with various cre-elements in Bacillus subtilis = Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis |
| title_full | Interactions of TCA cycle enzymes and of the CcpA-HPrSer46P complex with various cre-elements in Bacillus subtilis = Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis von Maike Bartholomae |
| title_fullStr | Interactions of TCA cycle enzymes and of the CcpA-HPrSer46P complex with various cre-elements in Bacillus subtilis = Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis von Maike Bartholomae |
| title_full_unstemmed | Interactions of TCA cycle enzymes and of the CcpA-HPrSer46P complex with various cre-elements in Bacillus subtilis = Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis von Maike Bartholomae |
| title_short | Interactions of TCA cycle enzymes and of the CcpA-HPrSer46P complex with various cre-elements in Bacillus subtilis |
| title_sort | interactions of tca cycle enzymes and of the ccpa hprser46p complex with various cre elements in bacillus subtilis interaktionen von enzymen des citratzyklus und des ccpa hprser46p komplexes mit verschiedenen cre elementen in bacillus subtilis |
| title_sub | = Interaktionen von Enzymen des Citratzyklus und des CcpA-HPrSer46P-Komplexes mit verschiedenen cre-Elementen in Bacillus subtilis |
| topic | Heubacillus (DE-588)4242465-3 gnd Genregulation (DE-588)4122166-7 gnd Citronensäurezyklus (DE-588)4148058-2 gnd Enzymaktivität (DE-588)4152470-6 gnd |
| topic_facet | Heubacillus Genregulation Citronensäurezyklus Enzymaktivität Hochschulschrift |
| url | https://open.fau.de/handle/openfau/3367 https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-46498 http://d-nb.info/1035000814/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026106421&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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