Functional genome analysis in Pseudomonas aeruginosa SG17M [Elektronische Ressource] / von Anna Silke Limpert

Anna Silke Limpert

Sujets

• Biologie

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2005
• Nombre de lectures: 44
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

Functional Genome Analysis in

Pseudomonas aeruginosa SG17M


Vom Fachbereich Chemie der Universität Hannover
zur Erlangung des Grades
Doktorin der Naturwissenschaften
- Dr. rer. nat. -

genehmigte Dissertation

von

Dipl. Biochemikerin
Anna Silke Limpert



geboren am 02.08.1973 in Düsseldorf

Hannover 2005


















Die vorliegende Arbeit wurde in der Zeit vom 1.12.1999 bis zum 15.8.2004 in der
Klinischen Forschergruppe "Molekulare Pathologie der Mukoviszidose", Zentrum
Biochemie und Zentrum Kinderheilkunde der Medizinischen Hochschule Hannover unter
der Leitung von Prof. Dr. Dr. B.Tümmler angefertigt.






Tag der Promotion: 14. 12. 2004


Referent: Prof. Dr. Dr. B. Tümmler
Zentrum Biochemie und Zentrum Kinderheilkunde
Medizinische Hochschule Hannover


Korreferent: Prof. Dr. G.F. Gerlach
Institut für Mikrobiologie und Tierseuchen
Tierärztliche Hochschule Hannover
Table of Contents________________________________________________________________________________

Abstract ________________________________________________________________________ 4
Kurzzusammenfassung ____________________________________________________________ 5
1. Introduction __________________________________________________________________ 6
1.1. Pseudomonas aeruginosa ______________________________________________________ 6
1.1.1. P.aeruginosa SG17M and TB __________________________________________________ 7
1.1.2. Genome Organization and Functional Genomics in P.aeruginosa_______________________ 7
1.1.3. P.aeruginosa virulence, Cytotoxicity and Cystic Fibrosis ______________________________ 9
1.1.4. Quorum Sensing in P.aeruginosa ______________________________________________ 11
1.2. Methods for functional genomics in bacteria________________________________________ 13
1.2.1. Signature - Tagged Transposon Mutagenesis _____________________________________ 14
1.2.2. Directed Mutagenesis via Allelic Replacement ____________________________________ 16
1.2.3. DNA chip technology ________________________________________________________ 17
1.2.3.1. Expression analyses using DNA microarrays 17
1.2.3.2. Mutational analysis applying DNA SNP-chips 17
1.3. Objectives __________________________________________________________________ 18
2. Materials and Methods 19
2.1. Bacteria____________________________________________________________________ 19
2.2. Vectors 19
2.3. Equipment 22
2.4. Chemicals and Enzymes_______________________________________________________ 22
2.5. Consumables _______________________________________________________________ 22
2.6. Buffers and Solutions _________________________________________________________ 23
2.2.Microbiological Methods________________________________________________________ 26
2.2.1. Bacteria culture ____________________________________________________________ 26
2.2.2. Determination of the bacterial number ___________________________________________ 26
2.2.3. Longtime storage of bacteria __________________________________________________ 26
2.2.4. Antibiogram 26
2.3. Cell culture methods __________________________________________________________ 27
2.3.2. Culture of Monocytes and Differentiation into Macrophages __________________________ 27
2.4. Construction of an STM transposon library in P.aeruginosa SG17M _____________________ 27
2.4.1. Conjugation- Triparental Mating________________________________________________ 28
2.4.1.1. Pretreatment of the acceptor strain____________________________________________ 28
2.4.1.2. Optimization of the triparental mating __________________________________________ 28
2.4.2. Selection of the mutants______________________________________________________ 28
2.4.3. Quality assessment of the mutant library _________________________________________ 29
2.4.4. Arraying the mutants ________________________________________________________ 29
2.4.5. Southern Blotting to evaluate the mutagenesis efficiency ____________________________ 29
2.5. Bioassays __________________________________________________________________ 29
2.5.1. Assaying Quorum Sensing Capabilities of the mutants ______________________________ 29
2.5.2. Homoserine lactone (HSL) production assay______________________________________ 30
2.5.3.Qualitative assessment of Siderophore Production _________________________________ 30
1Table of Contents________________________________________________________________________________


2.5.4. Assessment of Biofilm Production ______________________________________________ 31
2.5.5. Establishing a Cytotoxicity Assay using THP-1 monocyte-derived macrophages __________ 31
2.5.4.1. Ethidium Bromide Staining as an indicator of cell viability loss _______________________ 31
2.5.4.2. MTT assay to assess cell viability _____________________________________________ 31
2.5.4.3. High-thoughput screening of the mutant library __________________________________ 32
2.6. Molecular Biology Methods _____________________________________________________ 32
2.6.1. Preparation of Genomic DNA from P.aeruginosa 32
2.6.2. Preparation of Genom P. aeruginosa for Direct Genomic Sequencing_________ 32
2.6.3. Y-linker PCR product generation and sequencing 33
2.6.4. Establishing an easy to use-protocol for directed mutagenesis employing gene replacement 34
2.6.4.1. Construction of the deletion sequence _________________________________________ 35
2.6.4.2. Polymerase Chain Reaction (PCR)____________________________________________ 35
2.6.4.2.1. Primer Design __________________________________________________________ 35
2.6.4.2.2. PCR Protocols 36
2.6.4.2.3. Purification of PCR products _______________________________________________ 36
2.6.5. Ligation___________________________________________________________________ 37
2.6.6. Subcloning procedures ______________________________________________________ 37
2.7. Transformation of chemically competent cells ______________________________________ 38
2.8. Preparation of Plasmid DNA ____________________________________________________ 38
2.9. Restriction analysis of plasmid- and genomic DNA___________________________________ 38
2.10. Analysis of DNA fragments by agarose gel electrophoresis ___________________________ 39
2.11. Recovery of DNA fragments from agarose gels ____________________________________ 39
2.12. Vector dephosphorylation _____________________________________________________ 40
2.13. Electroporation _____________________________________________________________ 40
2.14. DNA Analysis by Southern Hybridization _________________________________________ 41
2.14.1.DNA -Transfer onto Nylon Membranes__________________________________________ 41
2.14.2. Generation of DIG-Labeled Hybridization Probes _________________________________ 41
2.14.3. Random-Primed Labeling____________________________________________________ 41
2.14.4. DNA Fixation and Hybridization of specific DNA probes to a Southern blot______________ 41
2.14.5. Washing and Labeling of the Southern Blot______________________________________ 41
2.14.6. Labeling Reaction _________________________________________________________ 42
2.14.7. Immunological Detection of Digoxygenin-Labeled DNA_____________________________ 42
2.14.7. Washing and Stripping of Hybridized Membranes _________________________________ 42
3. Results and Discussion________________________________________________________ 43
3.1. Genotypic and phenotypic comparison of two highly virulent P.aeruginosa isolates _________ 43
3.1.1. Description of the respective phenotypes: ________________________________________ 44
3.1.2. Establishing the genotypic relationships between CHA and TB________________________ 44
3.1.3. Comparative sequencing of selected genes of CHA and PAOI 45
3.1.4. Electron microscopy monitoring intracellular survival of CHA and TB in fresh human PMN __ 47
3.1.5. Cytotoxicity of CHA and PMN towards human THP-1 macrophages____________________ 52
2Table of Contents________________________________________________________________________________

3.1.6. TTSS dependent killing of C.elegans____________________________________________ 54
3.1.7. Summary and Conclusion ____________________________________________________ 54
3.2. Construction of a Signature Tagged Mutant library in P.aeruginosa SG17M _______________ 55
3.2.1. Optimization of acceptor strain pre-treatment _____________________________________ 55
3.2.2. Optimization of the triparental mating 56
3.2.3. Arraying the mutants ________________________________________________________ 56
3.2.4. Quality control of the mutant library _____________________________________________ 57
3.3. Identifying and sequencing of genes with a transposon insertion ________________________ 58
3.3.1. Direct Genomic Sequencing __________________________________________________ 59
3.3.2 Y-linker method _____________________________________________________________ 59
3.4 Screening of the TB STM library for genes involved in cytotoxicity towards human monocyte-
derived macrophages 60
3.