Molecular characterization of cereulide synthesis in emetic Bacillus cereus [Elektronische Ressource] / Genia Lücking

Genia

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 45
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Mikrobielle Ökologie



Molecular characterization of cereulide synthesis
in emetic Bacillus cereus


GENIA LÜCKING


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. S. Scherer
Prüfer der Dissertation: 1. Univ.-Prof. Dr. M. Ehling-Schulz
(Veterinärmedizinische Universität Wien/Österreich)
2. Univ.-Prof. Dr. R. F. Vogel



Die Dissertation wurde am 27.01.2009 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 10.03.2009 angenommen.





























“If you don't make mistakes, you're not working on hard enough problems.
And that's a big mistake.”

Frank Wilczek
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 Pathogenicity of the Bacillus cereus group.................................................1
1.2 Cereulide and its synthetase gene cluster..................................................2
1.3 Regulation of virulence gene expression....................................................4
1.4 The Spo0A regulon.....................................................................................5
1.5 Research objective.....................................................................................7
2 Material and Methods.................................................................................8
2.1 Bacterial strains..........................................................................................8
2.2 Growth conditions.......................................................................................9
2.3 DNA isolation..............................................................................................9
2.4 RNA isolation............................................................................................10
2.5 Reverse transcription (RT-PCR)...............................................................10
2.6 Standard PCR ..........................................................................................11
2.7 Quantitative real time PCR (qPCR) ..........................................................11
2.8 DNA Sequencing......................................................................................12
2.9 Enzymatic modification of DNA ................................................................13
2.10 Transformation of DNA.............................................................................13
2.11 Heterogramic conjugation.........................................................................14
2.12 Mutagenesis of B. cereus .........................................................................14
2.12.1 Construction of B. cereus deletion mutants ..............................................16
2.12.2 Complementation of deletion mutants ......................................................17
2.12.3 Construction of B. cereus insertion mutants .............................................17
2.12.4 Construction of B. cereus strains overexpressing AbrB and AbrB* ..........18
2.12.5 Plasmid curing..........................................................................................18
2.13 Ces-promoter fusions and luciferase assay..............................................18
2.14 Overexpression and purification of AbrB and AbrB*.................................19
I Table of Contents

2.15 Gel mobility shift assay.............................................................................20
2.16 Cell fractionation and cereulide extraction................................................20
2.17 Cereulide toxicity HEp-2 cell assay ..........................................................21
2.18 Sample preparation for proteome analysis ...............................................22
2.19 Two-dimensional gel electrophoresis .......................................................22
2.20 Biofilm microtitre plate assay....................................................................23
3 Results .....................................................................................................24
3.1 Regulation of cereulide synthesis.............................................................24
3.1.1 Construction and phenotypic characterization of B. cereus deletion
mutants.....................................................................................................24
3.1.2 Cytotoxicity of B. cereus deletion mutants................................................26
3.1.3 Transcriptional analysis of cesA by RT-qPCR..........................................27
3.1.4 Spo0A complementation and AbrB overexpression .................................28
3.1.5 ces promoter studies ................................................................................31
3.1.6 In vitro binding of AbrB and AbrB*............................................................32
3.1.7 Characterization of insertion mutants concerning the regulator genes sigH,
codY and ccpA .........................................................................................35
3.1.8 Effect of glucose and other external factors on cereulide synthesis .........37
3.2 Functional analysis of ces locus genes ....................................................42
3.2.1 Role of the phosphopantetheinyl transferases CesP and Ppt on cereulide
formation ..................................................................................................42
3.2.2 Cereulide production of cesH- and cesCD deficient mutants ...................44
3.2.3 Transcription of cesA in B. cereus ces mutant strains..............................45
3.2.4 In silico analysis of CesCD .......................................................................46
3.2.5 Localization of cereulide in vegetative cells..............................................47
3.3 Characterization of the megaplasmid pBCE.............................................50
3.3.1 Plasmid curing of B. cereus F4810/72......................................................50
3.3.2 Proteomic analysis of B. cereus mutants lacking pBCE ...........................51
3.3.3 Biofilm studies ..........................................................................................53
4 Discussion ................................................................................................55
4.1 Regulation of cereulide expression in B. cereus is complex.....................55
4.1.1 Cereulide synthesis depends on Spo0A-AbrB regulon, but not on PlcR ..55
IITable of Contents

H
4.1.2 Role of the alternative sigma factor and the regulatory factors CodY
and CcpA..................................................................................................57
4.1.3 Effect of glucose and other extrinsic factors influencing cereulide
production.................................................................................................60
4.2 Functional characterization of ces genes and the megaplasmid pBCE....62
4.2.1 Impact of cesH and cesP on cereulide synthesis .....................................62
4.2.2 Putative ABC transporter CesCD is essential for cereulide production ....64
4.2.3 Toxin plasmid pBCE influences secretome and biofilm formation ............65
4.3 Conclusion and perspectives....................................................................67
5 References ...............................................................................................69
6 Publications ..............................................................................................81
7 Appendix ..................................................................................................83
7.1 Vectors and recombinant plasmids...........................................................83
7.2 Oligonucleotide primers............................................................................85
Acknowledgements ...................................................................................................91
Curriculum