Molecular characterization of the genetic locus responsible for cereulide toxin production in emetic Bacillus cereus [Elektronische Ressource] / Monica Karen Dommel

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 37
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

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TECHNISCHE UNIVERSITÄT MÜNCHEN
LEHRSTUHL FÜR MIKROBIELLE ÖKOLOGIE




Molecular characterization of the genetic locus
responsible for cereulide toxin production in
emetic Bacillus cereus


MONICA KAREN DOMMEL



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. A. Gierl
Prüfer der Dissertation: 1. Univ.-Prof. Dr. S. Scherer
2. Univ.-Prof. Dr. E. Märtlbauer
(Ludwig-Maximilians-Universität München)


Die Dissertation wurde am 06.02.2008 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 29.04.2008 angenommen.

























“There are some things so serious, you have to laugh at them.”
~Niels Bohr~
Monica K Dommel Table of Contents
Table of Contents
TABLE OF CONTENTS............................................................................................................I
LIST OF FIGURES.................................................................................................................IV
LIST OF TABLES ..................................................................................................................VI
SUMMARY............................................................................................................................ VII
ZUSAMMENFASSUNG....................................................................................................... VIII
1 INTRODUCTION...............................................................................................................1
1.1 THE BACILLUS CEREUS GROUP.....................................................................................1
1.2 EMETIC BACILLUS CEREUS...........................................................................................2
1.3 NON-RIBOSOMAL PEPTIDE SYNTHESIS (NRPS) .............................................................3
1.4 RESEARCH OBJECTIVE.................................................................................................5
2 MATERIALS AND METHODS .........................................................................................6
2.1 BACTERIAL STRAINS AND CULTURE CONDITIONS............................................................6
2.1.1 Bacterial strains ..................................................................................................6
2.1.2 Culture conditions...............................................................................................6
2.2 CEREULIDE TOXICITY ASSAY8
2.3 DNA ISOLATION...........................................................................................................9
2.4 POLYMERASE CHAIN REACTION (PCR) .........................................................................9
2.5 CLONING AND SEQUENCING .......................................................................................10
2.5.1 TOPO TA cloning..............................................................................................10
2.5.2 Fluorescent IRD800 dye sequencing................................................................10
2.6 RNA PURIFICATION. ..................................................................................................10
2.6.1 RNA Isolation....................................................................................................10
2.6.2 DNA Digestion11
2.7 REVERSE TRANSCRIPTION PCR (RT-PCR)11
2.8 RAPID AMPLIFICATION OF CDNA ENDS (5’RACE)........................................................12
2.9 PRIMER EXTENSION ...................................................................................................14
2.10 TRANSCRIPT STABILITY...........................................................................................14
2.11 BIO-INFORMATICS ANALYSIS ...................................................................................14
2.12 CONSTRUCTION OF TRANSCRIPTIONAL FUSIONS......................................................15
2.12.1 Fusion vector construction................................................................................15
2.12.2 Bacterial transformation....................................................................................16
2.12.3 Confirmation of correct ligation.........................................................................16
2.13 GREEN FLUORESCENT PROTEIN ASSAY ...................................................................17
I Monica K Dommel Table of Contents
2.14 LUCIFERASE ASSAY................................................................................................17
2.15 QUANTITATIVE REAL TIME PCR...............................................................................18
2.15.1 Primer design....................................................................................................18
2.15.2 Reverse transcription........................................................................................18
2.15.3 Quantitative real time PCR19
2.15.4 Relative expression calculation ........................................................................20
3 RESULTS .......................................................................................................................21
3.1 TRANSCRIPT CHARACTERIZATION ...............................................................................21
3.1.1 Cereulide synthetase genes a polycistronic transcript......................................21
3.2 PROMOTER CHARACTERIZATION.................................................................................24
3.2.1 Central and intercistronic promoters detected ..................................................24
3.2.2 Sequence analysis............................................................................................28
3.2.3 Deletion analysis shows strong main promoter ................................................28
3.2.4 Comparison of highly and weakly emetic strains..............................................32
3.2.5 Promoter activity in supplemented media.........................................................35
3.2.6 Promin media and foods................................................................38
3.3 TRANSCRIPTIONAL KINETICS.......................................................................................41
3.3.1 SYBR Green I assay.........................................................................................41
3.3.2 Growth-phase dependent expression independent of plcR levels....................43
3.3.3 Lower expression of weakly emetic strain ........................................................48
3.3.4 Sporulation deficient mutant shows depressed ces expression .......................48
3.3.5 Emetic B. weihenstephanensis shows drastically reduced expression ............49
3.3.6 Environmental effects51
4 DISCUSSION..................................................................................................................57
4.1 4’-PHOSPHOPANTETHEINYL TRANSFERASE INTEGRAL TO CEREULIDE NRPS ................57
4.2 PROMOTER STRENGTH CANNOT ACCOUNT FOR TOXICITY DIFFERENCES.......................59
4.3 REGULATION APPEARS TO BE CHROMOSOMAL BUT INDEPENDENT OF THE CENTRAL
VIRULENCE REGULATOR PLCR...............................................................................................60
4.4 IMPACT OF SPORULATION...........................................................................................63
4.5 EFFECT OF CULTURE CONDITIONS ON CEREULIDE SYNTHETASE GENE EXPRESSION AND
TOXIN LEVELS.......................................................................................................................65
5 CONCLUSION AND PERSPECTIVES...........................................................................69
6 REFERENCES................................................................................................................70
7 APPENDIX......................................................................................................................81
7.1 BACILLUS STRAINS.....................................................................................................81
7.2 OLIGONUCLEOTIDE PRIMERS......................................................................................82
II Monica K Dommel Table of Contents
7.3 PLASMIDS..................................................................................................................84
7.4 GENETICALLY MODIFIED ORGANISMS ..........................................................................85
8 ACKNOWLEDGEMENTS...............................................................................................86
CURRICULUM VITAE ...........................................................................................................87
III Monica K Dommel List of Figures
List of Figures
Figure 1. Arrangement of t