Regulation of the biosynthesis of the food-borne Bacillus cereus toxin cereulide:
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
2011
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| Subjects: | |
| Online Access: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20111027-1078757-0-7 Inhaltsverzeichnis |
| Item Description: | Zsfassung in dt. Sprache |
| Physical Description: | X, 136 S. Ill., graph. Darst. |
Staff View
MARC
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| 100 | 1 | |a Frenzel, Elrike |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Regulation of the biosynthesis of the food-borne Bacillus cereus toxin cereulide |c Elrike Frenzel |
| 264 | 1 | |c 2011 | |
| 300 | |a X, 136 S. |b Ill., graph. Darst. | ||
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| 337 | |b n |2 rdamedia | ||
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| 502 | |a München, Techn. Univ., Diss., 2011 | ||
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| 650 | 0 | 7 | |a Regulation |0 (DE-588)4049075-0 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Bacillus cereus |0 (DE-588)4462648-4 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Bacillus cereus |0 (DE-588)4462648-4 |D s |
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Record in the Search Index
| _version_ | 1804150159036121088 |
|---|---|
| adam_text | IMAGE 1
TABLE O F CONTENTS
CONTENTS
CONTENTS I
PUBLICATIONS IV
SUMMARY V
ZUSAMMENFASSUNG VII
ABBREVIATION AND SYMBOL INDEX IX
1 INTRODUCTION AND RESEARCH OBJECTIVES 1 -
1 . 1 BACILLUS CEREUS AND ITS CLOSEST RELATIVES 1
1.2 PATHOGENICITY O F B. CEREUS 2
1.3 REGULATION O F VIRULENCE FACTOR EXPRESSION IN B. CEREUS 5
1.3.1 TRANSITION AND STATIONARY PHASE VIRULENCE FACTORS 5
1.3.2 CEREULIDE AND ITS BIOSYNTHETIC GENES 6
1.4 RESEARCH OBJECTIVES 9
2 MATERIALS AND METHODS 11
2. 1 BACTERIAL STRAINS AND PLASMIDS 11
2.2 MEDIA AND GROWTH CONDITIONS 13
2.3 CHEMICALS 15
2.4 FOOD MICROBIOLOGY TECHNIQUES 16
2.4.1 MPN METHOD FOR ENUMERATION O F PRESUMPTIVE B. CEREUS 16
2.4.2 DETECTION O F EMETIC B. CEREUS BY STANDARD AND MULTIPLEX PCR 17
2.4.3 ENRICHMENT O F B. CEREUS IN FOOD SAMPLES 17
2.4.4 DNA EXTRACTION FROM FOOD AND TAQMAN REAL-TIME PCR 18
2.4.5 ARTIFICIAL INOCULATION AND GROWTH O F B. CEREUS IN FOODS 18
2.4.6 EXTRACTION O F FOODS FOR CEREULIDE QUANTIFICATION 19
2.4.7 CEREULIDE QUANTIFICATION (HPLC/ESI-TOF-MS) 19
2.4.8 REPORTER GENE ANALYSIS O F CES PROMOTER ACTIVITY IN MODEL FOODS 20
2.5 COMPUTATIONAL TECHNIQUES / IN SILICO ANALYSES 20
2.6 NUCLEIC ACID TECHNIQUES 21
2.6.1 ISOLATION O F PLASMID DNA FROM E. COLI 21
2.6.2 ISOLATION O F GENOMIC DNA FROM BACILLUS SPP 21
2.6.3 STANDARD PCR 21
2.6.4 DNA AGAROSE GEL ELECTROPHORESIS 22
2.6.5 ENZYMATIC MODIFICATION O F DNA 2 3
2.6.6 PURIFICATION AND CONCENTRATION O F DNA FRAGMENTS 2 3
HTTP://D-NB.INFO/1033872938
IMAGE 2
TABLE O F CONTENTS
2.6.7 ISOLATION O F TOTAL RNA FROM B. CEREUS 2 3
2.6.8 REVERSE TRANSCRIPTION (RT-PCR) 2 3
2.6.9 QUANTITATIVE REAL-TIME PCR (QPCR) 24
2.7 TRANSFORMATION AND MUTAGENESIS O F BACTERIA 2 6
2.7.1 TRANSFORMATION O F E. COLI 2 6
2.7.2 TRANSFORMATION O F B. CEREUS 2 6
2.7.3 ALLELIC GENE REPLACEMENT BY HETEROGRAMIC CONJUGATION 2 6
2.7.4 CONSTRUCTION O F E. COLI PROTEIN EXPRESSION STRAINS 2 7
2.7.5 CONSTRUCTION O F A B. CEREUS CODY DELETION MUTANT 2 9
2.7.6 CONSTRUCTION O F A B. CEREUS CODY OVEREXPRESSION STRAIN 2 9
2.7.7 CONSTRUCTION O F CAS-LUCIFERASE REPORTER STRAINS 2 9
2.7.8 CONSTRUCTION O F A CES-GFP REPORTER STRAIN 30
2.8 PROTEIN BIOCHEMISTRY TECHNIQUES 31
2.8.1 PROTEIN QUANTIFICATION 31
2.8.2 PREPARATION O F CYTOSOLIC PROTEIN FRACTIONS FOR WESTERN BLOT
ANALYSIS 3 2
2.8.3 PRE-FRACTIONING O F CYTOSOLIC AND SECRETED PROTEOMES FOR 2-D DIGE
3 2
2.8.4 RECOMBINANT PROTEIN EXPRESSION AND PURIFICATION 33
2.8.5 GEL MOBILITY SHIFT ASSAY 34
2.8.6 DENATURING PROTEIN SEPARATION (SDS-PAGE) 37
2.8.7 WESTERN BLOT 38
2.8.8 TWO-DIMENSIONAL DIFFERENTIAL GEL ELECTROPHORESIS (2-D DIGE) 38
2.8.9 PROTEIN IDENTIFICATION BY MALDI-TOF-MS(MS) 40
2.9 METHODS FOR VIRULENCE FACTOR DETECTION 41
2.9.1 CEREULIDE QUANTIFICATION (HEP-2 CELL ASSAY) 41
2.9.2 ENTEROTOXIN QUANTIFICATION (VERO CELL ASSAY) 4 2
2.9.3 PHOSPHATIDYLCHOLINE-SPECIFIC PHOSPHOLIPASE C (PC-PLC) ASSAY AND
SPHINGOMYELINASE (SPH) ASSAY 4 2
2.9.4 HORSE RED BLOOD CELL (HRBC) HEMOLYSIS ASSAY 4 3
3 RESULTS 45
3.1 DETECTION O F EMETIC B. CEREUS AND CEREULIDE IN FOODS 4 5
3.2 EVALUATION O F A PI-LUX REPORTER STRAIN AS A RAPID SCREENING TOOL
FOR CEREULIDE SYNTHESIS IN FOODS 4 7
3.3 INHIBITION O F CEREULIDE SYNTHESIS BY LONG-CHAIN POLYPHOSPHATES 52
3.3.1 INFLUENCE O F POLYPS ON CEREULIDE SYNTHESIS IN MODEL FOODS 52
3.3.2 INFLUENCE O F POLYPS ON CEREULIDE SYNTHESIS UNDER DEFINED
CONDITIONS 55
3.4 IDENTIFICATION O F TRANSCRIPTION FACTORS INVOLVED IN REGULATION O F
CES NRPS EXPRESSION 58
3.4.1 IN SILICO BINDING PREDICTION AND BEST RECIPROCAL HIT ANALYSIS 59
II
IMAGE 3
TABLE O F CONTENTS
3.4.2 IN VITRO CES PROMOTER BINDING ANALYSES 6 2
3.5 THE ROLE O F CODY IN VIRULENCE GENE REGULATION O F EMETIC B. CEREUS
6 6
3.5.1 INFLUENCE O F CODY ON CEREULIDE SYNTHESIS 6 6
3.5.2 INFLUENCE O F CODY ON TRANSITION AND STATIONARY PHASE VIRULENCE
FACTORS.70
3.6 SUBPROTEOME ANALYSIS O F B. CEREUS F4810/72 AND ITS DERIVATIVES 74
3.7 CELL POPULATION HETEROGENEITY IN B. CEREUS F4810/72 82
4 DISCUSSION 87
4.1 EMETIC B. CEREUS: EMERGING FOODBORNE PATHOGENS 87
4.1.1 INCIDENCE O F EMETIC B. CEREUS AND CEREULIDE IN DAIRY-BASED FOODS
87
4.1.2 A /UX-BASED REPORTER STRAIN AS NOVEL TOOL TO ENHANCE B. CEREUS
FOOD SAFETY 88
4.1.3 INITIAL STEPS TOWARDS TARGETED HURDLE CONCEPTS IN B. CEREUS
FOOD SAFETY 90
4.2 CEREULIDE SYNTHESIS: REGULATION AT THE INTERSECTION O F METABOLISM
AND
VIRULENCE 92
4.2.1 CELLULAR PHOSPHATE STATUS IS CRUCIAL FOR CEREULIDE SYNTHESIS 92
4.2.2 A NETWORK O F REGULATORS FINE-TUNES TRANSCRIPTION O F THE CES
OPERON 94
4.2.3 CODY: A CENTRAL REGULATOR O F VIRULENCE 98
4.2.3.1 CODY REPRESSES CEREULIDE TOXIN SYNTHESIS 9 8
4.2.3.2 CODY ACTIVATES TRANSITION AND STATIONARY PHASE VIRULENCE FACTORS
100
4.2.4 PLASMID-CHROMOSOME CROSSTALK IN B. CEREUS F4810/72 102
4.2.5 CEREULIDE SYNTHESIS IS A BISTABLE PHENOMENON 108
5 CONCLUDING REMARKS AND PERSPECTIVES 113
6 REFERENCES 115
7 APPENDIX 133
III
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| spelling | Frenzel, Elrike Verfasser aut Regulation of the biosynthesis of the food-borne Bacillus cereus toxin cereulide Elrike Frenzel 2011 X, 136 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. Sprache München, Techn. Univ., Diss., 2011 Biosynthese (DE-588)4006902-3 gnd rswk-swf Lebensmitteluntersuchung (DE-588)4034911-1 gnd rswk-swf Toxin (DE-588)4127018-6 gnd rswk-swf Regulation (DE-588)4049075-0 gnd rswk-swf Bacillus cereus (DE-588)4462648-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Bacillus cereus (DE-588)4462648-4 s Toxin (DE-588)4127018-6 s Biosynthese (DE-588)4006902-3 s Regulation (DE-588)4049075-0 s Lebensmitteluntersuchung (DE-588)4034911-1 s b DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:91-diss-20111027-1078757-0-7 http://mediatum.ub.tum.de/node?id=1078757 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20111027-1078757-0-7 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025857377&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Frenzel, Elrike Regulation of the biosynthesis of the food-borne Bacillus cereus toxin cereulide Biosynthese (DE-588)4006902-3 gnd Lebensmitteluntersuchung (DE-588)4034911-1 gnd Toxin (DE-588)4127018-6 gnd Regulation (DE-588)4049075-0 gnd Bacillus cereus (DE-588)4462648-4 gnd |
| subject_GND | (DE-588)4006902-3 (DE-588)4034911-1 (DE-588)4127018-6 (DE-588)4049075-0 (DE-588)4462648-4 (DE-588)4113937-9 |
| title | Regulation of the biosynthesis of the food-borne Bacillus cereus toxin cereulide |
| title_auth | Regulation of the biosynthesis of the food-borne Bacillus cereus toxin cereulide |
| title_exact_search | Regulation of the biosynthesis of the food-borne Bacillus cereus toxin cereulide |
| title_full | Regulation of the biosynthesis of the food-borne Bacillus cereus toxin cereulide Elrike Frenzel |
| title_fullStr | Regulation of the biosynthesis of the food-borne Bacillus cereus toxin cereulide Elrike Frenzel |
| title_full_unstemmed | Regulation of the biosynthesis of the food-borne Bacillus cereus toxin cereulide Elrike Frenzel |
| title_short | Regulation of the biosynthesis of the food-borne Bacillus cereus toxin cereulide |
| title_sort | regulation of the biosynthesis of the food borne bacillus cereus toxin cereulide |
| topic | Biosynthese (DE-588)4006902-3 gnd Lebensmitteluntersuchung (DE-588)4034911-1 gnd Toxin (DE-588)4127018-6 gnd Regulation (DE-588)4049075-0 gnd Bacillus cereus (DE-588)4462648-4 gnd |
| topic_facet | Biosynthese Lebensmitteluntersuchung Toxin Regulation Bacillus cereus Hochschulschrift |
| url | http://mediatum.ub.tum.de/node?id=1078757 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20111027-1078757-0-7 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025857377&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT frenzelelrike regulationofthebiosynthesisofthefoodbornebacilluscereustoxincereulide |