Facultad de Ciencias Departamento de Biología Molecular Programa de Doctorado en Microbiología pSM19035: dissection of the plasmid partitioning machinery –TESIS DOCTORAL– Memoria presentada para optar al grado de Doctor en Ciencias Andrea Volante TUTOR ACADÉMICO: DIRECTOR: Silvia Ayora Juan Carlos Alonso Silvia Ayora Consejo Superior de Investigaciones Científicas Centro Nacional de Biotecnología Madrid, 2014 Contents Contents.................................................................................................................................... III Index of Figures ...................................................................................................................... VII Index of Tables ......................................................................................................................... IX Abbreviations ............................................................................................................................ XI Abstract ....................................................................................................................................... 1 Introduction ................................................................................................................................ 5 1. Low–copy Streptococcus pyogenes pSM19035 plasmid. ...................................................... 7 1.1. The segA locus maximizes random segregation. ................................................................ 9 1.2. The segB loci promote better–than–random segregation. ................................................ 10 1.2.1. The segB1 encodes for a toxin–antitoxin system. ........................................................ 10 1.2.2. The segB2 encode for active partition system. ............................................................ 12 1.3. The segC locus synchronizes leading and lagging strand replication and avoids a replication fork collapse.................................................................................................... 13 1.4. The segD locus modulates the interplay between copy number fluctuation and plasmid better–than–random segregation. ...................................................................................... 13 2. Active partition systems. ...................................................................................................... 16 2.1. Type I par system. ............................................................................................................. 18 2.1.1. Bacterial chromosomal par system. ............................................................................. 21 2.1.2. The pSM19035 par system. ......................................................................................... 22 2.2. Type II par system. ........................................................................................................... 24 2.3. Type III par system. .......................................................................................................... 26 3. Repression of transcription initiation in Bacteria. ............................................................ 26 3.1. Bacterial RNAP. ............................................................................................................... 27 3.2. Transcription initiation at bacterial promoter ................................................................... 29 3.3. Regulation of Transcription initiation............................................................................... 30 3.4. The RHH superfamily of transcription factors ................................................................. 31 Objectives .................................................................................................................................. 37 Materials and Methods ............................................................................................................ 41 1. Materials ............................................................................................................................... 43 1.1. Strains. .............................................................................................................................. 43 1.2. Plasmids. ........................................................................................................................... 43 1.3. Reagents and Materials. .................................................................................................... 47 III 1.4. Primers. ............................................................................................................................. 49 1.5. Media. ............................................................................................................................... 50 1.6. Buffers. ............................................................................................................................. 50 2. Methods ................................................................................................................................. 51 2.1. Cells manipulation. ........................................................................................................... 51 2.1.1. Competent cells production. ........................................................................................ 51 2.1.2. Bacterial Transformation. ............................................................................................ 51 2.2. Protein purification. .......................................................................................................... 52 2.2.1. Protein overproduction. ................................................................................................ 52 2.2.2. Protein purification. ..................................................................................................... 53 2.3. DNA manipulation. ........................................................................................................... 55 2.3.1. DNA isolation and quantification. ............................................................................... 55 2.3.2. DNA electrophoresis. ................................................................................................... 55 2.3.3. Radioactive DNA labelling. ......................................................................................... 55 2.3.4. Preparation and purification of DNA fragments. ......................................................... 56 2.3.5. Site–specific mutagenesis. ........................................................................................... 56 2.4. In vitro assays. .................................................................................................................. 56 2.4.1. Non–denaturing and denaturing protein electrophoresis. ............................................ 56 2.4.2. Homo– and hetero–oligomeric interactions between proteins. .................................... 58 2.4.3. Limited proteolysis assay. ............................................................................................ 58 2.4.4. Peptide mass fingerprinting MS. .................................................................................. 59 2.4.5. Western blotting analysis. ............................................................................................ 59 2.4.6. Far western blotting analysis........................................................................................ 59 2.4.7. Protein and DNA interactions. ..................................................................................... 60 2.4.8. Transcription run–off experiment. ............................................................................... 61 2.4.9. ATPase assay. .............................................................................................................. 62 2.4.10. Electron Microscopy. ................................................................................................... 63 2.5. In vivo assays. ................................................................................................................... 63 2.5.1. Plasmid stability. .......................................................................................................... 63 2.5.2. β–Galactosidase assays. ............................................................................................... 64 2.5.3. Fluorescence Microscopy. ........................................................................................... 64 Results ....................................................................................................................................... 65 1. Chapter I: The pSM19035 plasmid partition system. ....................................................... 67 1.1. Molecular characterization of pSM19035 partition system. ............................................. 67 IV 1.1.1. ω protein forms a discrete complex on parS DNA. .................................................... 67 2 1.1.2. Protein δ forms diffuse complex on DNA. ................................................................. 67 2 1.1.3. Proteins ω and δ bind parS DNA forming segresome and bridging complexes. ....... 69 2 2 1.1.4. The interaction of ω with δ markedly increases partition complex formation. ......... 69 2 2 1.1.5. Interaction of ω with δ discriminates between short–living and long–living partition 2 2 complexes. ............................................................................................................................. 70 1.1.6. Protein ω –bound to parS DNA promotes δ re–localization. ..................................... 71 2 2 1.1.7. Characterization of DNA binding domain of δ .......................................................... 73 2. 1.1.8.  – interacting domains. ........................................................................................... 84 2 2 1.2. Cytological characterization of pSM19035 par system. ................................................... 91 1.2.1. Quantification of ω and δ molecules per cell. ............................................................ 91 2 2 1.2.2. The δ DNA binding domain and its role in the plasmid stability. .............................. 92 2 1.2.3. Protein δ facilitates plasmid pairing. .......................................................................... 94 2 1.2.4. Protein ω bound to parS stimulates δ disassembles from the nucleoid. .................... 95 2 2 1.2.5. Nucleoid bound δ:CFP capture and tethers plasmid copies. ...................................... 98 2 1.2.6. The ω –parS complexes facilitate δ redistribution over the nucleoid. ....................... 98 2 2 2. Chapter II: Protein 2 regulates plasmid copy number, faithful partition and the toxin–antitoxin system. ...................................................................................................... 103 2.1. Molecular characterization of ω transcriptional regulation. .......................................... 103 2 2.1.1. RNAP–σA facilitates ω –P DNA interaction. .......................................................... 103 2 ω 2.1.2. RNAP–σA and ω2 can co–exist at P DNA region. ................................................... 105 2.1.3. RNAP–σA does not affect cooperative DNA binding of δ and ω ............................ 107 2 2 2.1.4. Protein ω affects P utilization. ................................................................................ 108 2 ω 2.1.5. Protein ω inhibits RNAP–σA transition from close to opencomplex. ...................... 109 2 2.1.6. Protein δ does not inhibits RNAP–σA transition from RP to RP complex. ....... 112 2 C INIT 2.1.7. Mapping ω –RNAP–σA domains. .............................................................................. 113 2 2.2. Characterization of ω2 transcriptional regulation. ........................................................ 116 2 2.2.1. Protein ω2 is a functional transcription repressor. .................................................... 116 2 2.2.2. Protein ω2 is functionally active in plasmid partition. ............................................. 117 2 2.3. Characterization of ω α1–α2 domains for the repression of P utilization. .................. 117 2 ω Discussion ................................................................................................................................ 119 1. The active partition systems of pSM19035. ..................................................................... 122 1.1. The role of δ C–terminal region is essential for pSM19035 segregation. ..................... 122 2 1.2. Protein ω interacts with the central domain of δ . ......................................................... 125 2 2 V 1.3. Molecular model explaining the pSM19035 plasmid partition system. ......................... 127 1.3.1. Formation of partition and dynamic complexes (PC1 and DC). ................................ 127 1.3.2. Dynamics of PC, SC and BC formation and disassembly. ........................................ 128 2. The global ω2 regulator...................................................................................................... 132 2.1. The dual behaviour of the ω regulator. .......................................................................... 133 2 2.2. Interplay between the regulator and ’ subunits of RNAP–. ....................................... 137 Conclusions ............................................................................................................................. 139 Acknowledgements ................................................................................................................. 143 Appendixes .............................................................................................................................. 147 1. List of Publications ............................................................................................................. 149 2. Playing with bioinformatics. .............................................................................................. 150 2.1. In silico analysis of ω and δ proteins. ........................................................................... 150 2 2 2.2. B. subtilis RNA polymerase: 3D structure prediction. ................................................... 153 3. Resumen en Castellano. ..................................................................................................... 156 3.1. Introducción ...................................................................................................................... 156 3.2. Objetivos ........................................................................................................................... 157 3.3. Resultados y Discusión ..................................................................................................... 157 3.4. Conclusiones. .................................................................................................................... 158 References ............................................................................................................................... 161 VI