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Table of contents
1 INTRODUCTION
1.1 General introduction
1.1.1 Project context
1.1.2 Bacillus subtilis, a Gram-positive bacterium
1.1.3 B. subtilis: a Gram-positive model
1.1.4 B. subtilis: a bacterium of industrial interest
1.2 Protein translocation across cytoplasmic membrane in bacteria
1.2.1 The general protein secretion pathway: the Sec system
1.2.2 Substrate recognition
1.2.3 Intracellular chaperoning and targeting to the Sec translocase
1.2.4 Twin-Arginine Transport (Tat) Pathway
1.2.5 Flagella Export Apparatus (FEA)
1.2.6 Type IV Secretion Systems
1.2.7 Holins
1.3 A guarantee of a correct protein translocation in B. subtilis
1.3.1 A bacterial cytoplasmic quality control system: ClpXP protease
1.3.2 Membrane-bound proteases, an important quality control step for membrane and secretory proteins.
1.3.3 Signal peptide peptidase A (SppA)
1.4 Scope of the Thesis
2 Results
2.1 Identification of membrane protein complexes involved in the B. subtilis Sec pathway
2.1.1 SPA-tag constructions
2.1.2 Purifications of the SPA-tagged proteins
2.1.3 Optimisation of the BN-PAGE technique
2.2 The SppA/YteJ membrane complex
2.2.1 The sppA yteJ operon is regulated by sigma factors σA and σW
2.2.2 Deletion of the sppA and yteJ genes
2.2.3 Role of the SppA/YteJ complex in protein secretion
2.2.4 The deletion of sppA yteJ affects the long term survival
2.2.5 The deletion of sppA and yteJ affects the resistance to lantibiotics
2.2.6 The deletions of sppA and yteJ do not affect the resistance to vancomycin, erythromycin or protect against lysozyme
2.2.7 Overexpression of sppA results in the recovery of the BSB1 phenotype in the presence of subtilin.
2.2.8 The overexpression of sppA results in a change of the cell morphology.
2.2.9 SppA and YteJ cell localization
2.2.10 SppA and YteJ in vitro purification.
2.2.11 SppA can digest fully folded proteins or not
2.2.12 YteJ inhibits SppA activity
2.2.13 The C-terminal domain of YteJ is involved in the regulation of SppA activity
2.2.14 SppA digests subtilin
3 Discussion and perspectives
3.1 Protein interactions of the B. subtilis Sec pathway
3.2 SppA/YteJ membrane protein complex
3.2.1 Role of SppA and YteJ in protein secretion:
3.2.2 Role in the resistance to nisin, subtilin and LP9
3.2.3 Regulation of the sppA yteJ operon
3.2.4 SppA/YteJ, a complex involved in quality control of cell division?
3.2.5 The protease activity of SppA and its regulation by YteJ
3.2.6 YteJ, another function?
3.2.7 Model for the interaction of SppA and YteJ
3.3 Main results
4 MATERIAL AND METHODS
4.1 Techniques for DNA manipulation
4.1.1 Oligonucleotide
4.1.2 Polymerase chain reaction (PCR)
4.1.3 Purification of PCR products
4.1.4 Electrophoresis of DNA
4.1.5 Purification of DNA from agarose gel
4.1.6 Purification of plasmid DNA
4.1.7 Chromosomal DNA extraction
4.1.8 Digestion of DNA with restriction enzymes
4.1.9 Ligation of DNA fragments
4.1.10 Gibson assembly technology
4.1.11 Ligation-Independent Cloning (LIC)
4.1.12 DNA sequencing
4.1.13 DNA transformation
4.2 Bacterial strains and growth conditions
4.3 Strain construction
4.3.1 SPA tagged strains construction
4.3.2 Deletion mutants
4.3.3 SppA, YteJ and SppA-YteJ overexpressing strains
4.3.4 Construction of the GFP Fusions
4.3.5 His- tag constructions
4.4 Fluorescence Microscopy
4.5 Live-cell array (LCA)
4.6 Bacteria survival test
4.7 Swimming and swarming capacity
4.8 Biofilm formation
4.9 Protein analysis
4.9.1 Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis (SDS PAGE gels)
4.9.2 Blue Native PAGE
4.9.3 Western blot analysis
4.9.4 Secreted proteins analysis
4.9.5 Sequential Peptide Affinity (SPA) purification
4.9.6 His-tag purification
4.9.7 Double tag purification
5 BIBLIOGRAPHY
