Faculté des Sciences Département de Biologie Moléculaire Laboratoire de Génétique et Physiologie bactérienne Promoteur : Laurence Van Melderen
Experimental evolution with a global regulator mutant
in Escherichia coli
Ethel Seyll
Thèse présentée en vue de l’obtention du grade de Docteur en Sciences (Biologie Moléculaire)
2
3
1. Introduction 6
1.1. The regulator CsrA 6
CsrA is a post-transcriptional regulator 6
1.1.1.
CsrA is a global regulator 8
1.1.2.
1.2. The Csr network 9
Building up the Csr network 9
1.2.1.
Expanding the Csr network 10
1.2.2.
Are the regulatory Csr/Rsm sRNAs redundant? 12
1.2.3.
1.3. Experimental evolution 13
Introduction 13
1.3.1.
Advantages and limitations 13
1.3.2.
Mutations 14
1.3.3.
Evolutionary dynamics of regulatory networks 14
1.3.4.
2. Objective 16
3. Results 18
3.1. Deletion of csrA in E. coli CFT073 18
3.2. Experimental evolution with a ΔcsrA mutant 19
3.3. Phenotypic characterization of the ΔcsrA mutants 19
3.5. CsrA and metabolism 30
3.6. Stress 32
3.7. Whole-genome sequencing 33
3.8. The csrA evolved 1 mutant case 35
4. Discussion and Perspectives 37
4.1. Metabolism and energy status 37
4.2. Social behavior – Biofilm formation 42
4.3. How to cope with csrA deletion? 43
5. Conclusion 49
6. Materials and Methods 51
6.1. Strains, plasmids, and culture conditions 51
6.2. Evolution experiment 51
6.3. Competition experiments 51
6.4. Motility assay 52
6.5. Adhesion assay 52
4
6.7. Total protein extraction 53
6.8. 2-dimensional differential gel electrophoresis (2D-DIGE) 54
6.9. Total RNA extraction 54
6.10. Real-time RT-qPCR analysis 55
6.11. Confocal fluorescence microscopy to assess membrane potential 55
6.12. Determination of intracellular ROS level 55
6.13. Genomic DNA isolation 56
6.14. Whole genome sequencing 56
6.15. Optical mapping 57
6.16. Cell fractionation 57
7. References 60
