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Stability, Placement and Interactions of the Divisome Components in Nucleoid-Deprived Escherichia Coli Cells

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Stability, Placement and Interactions of the Divisome Components in Nucleoid-Deprived Escherichia Coli Cells Universidad Autónoma de Madrid Facultad de Ciencias Departamento de Biología Molecular Stability, placement and interactions of the divisome components in nucleoid-deprived Escherichia coli cells TESIS DOCTORAL Memoria presentada para optar al grado de Doctor en Ciencias Manuel Pazos Don Pedro TUTOR ACADÉMICO: DIRECTORES: José Berenguer Carlos Miguel Vicente Muñoz Paolo Natale Consejo Superior de Investigaciones Científicas Centro Nacional de Biotecnología Madrid, 2013 Cover: Fluorescence microscopy image of CSR603 cells overproducing FtsZ*-VM Este trabajo se ha realizado en el Centro Nacional de Biotecnología (CNB-CSIC) bajo la dirección del Doctor Paolo Natale y del Profesor de Investigación Miguel Vicente Muñoz, y en la Universidad de Texas (Houston, EEUU) durante una estancia de 6 meses en el laboratorio del Profesor William Margolin. Ha sido financiado por el programa de Formación de Personal Investigador del Ministerio de Educación y Ciencia. Agradecimientos: - A Miguel, por darme la oportunidad y los medios para poder realizar esta tesis doctoral y haberme animado, ayudado y enseñado a ser mejor científico en estos años. - A Paolo, por todo el tiempo y el esfuerzo que ha dedicado a este proyecto ("trabajando codo con codo"), por tener siempre tiempo para explicarme y enseñarme algo nuevo cada día, por su incansable espíritu crítico, por sus consejos, y por guiarme y ayudarme a mejorar esta tesis. - To William Margolin for all the good advices and help since I was in his lab, and to all the people in his lab (Daniel, Heather, Jennifer, Jesús, Jenny, Kim and Tushar) for making me feel like at home and having a lot of fun there. - A todo el laboratorio del 217: - a Pilar y Mercedes, por su paciencia y sabiduría, no sólo científica, por ser el buffer que todo lo tampona y hacer que todos los días sean mejores. - a Cris, por toda la química que me ha ayudado a recordar y ser mi compi durante casi toda esta tesis. - a todos los demás que están o estuvieron (Moira, Anabel, Susanne, Marcin, Alicia, Marta, Javi, Magaly) por todo el tiempo que hemos pasado juntos en estos años, toda vuestra ayuda, las cosas que me habeis enseñado y todos los buenos momentos que hemos pasado. - A mis amigos de toda la vida porque ¿qué sería de la vida sin ellos? - Finalmente a mi familia, especialmente a mis padres y a mi hermano, por todo lo que no abarca esta tesis, porque sin ellos hubiera sido imposible empezarla, continuarla y acabarla. Contents CONTENTS Figure Index.............................................................................................................................................. 4 Table Index ............................................................................................................................................... 5 Abbreviations........................................................................................................................................... 7 Summary..................................................................................................................................................11 Introducción...........................................................................................................................................15 Introduction ...........................................................................................................................................29 1. Cell division process in Escherichia coli .............................................................................................31 2. Proto­ring assembly..................................................................................................................................32 2.1. FtsZ.............................................................................................................................................................................. 33 2.2. ZipA............................................................................................................................................................................. 34 2.3. FtsA ............................................................................................................................................................................. 36 3. Z­ring effector proteins............................................................................................................................37 3.1. Z‐ring stabilizing proteins................................................................................................................................. 38 3.2. Z‐ring inhibiting proteins.................................................................................................................................. 40 4. Septum placement macHinery...............................................................................................................42 4.1. Nucleoid occlusion ............................................................................................................................................... 42 4.2. Min system............................................................................................................................................................... 43 5. SyntHetic biology approacH ....................................................................................................................45 5.1. Maxicells ................................................................................................................................................................... 45 Objectives................................................................................................................................................47 Materials & Methods............................................................................................................................51 1. Materials .......................................................................................................................................................53 1.1. Escherichia coli strains ....................................................................................................................................... 53 1.2. Plasmids.................................................................................................................................................................... 54 1.3. Antisera..................................................................................................................................................................... 57 1.4. Primers...................................................................................................................................................................... 58 1.5. Chemical and reagents........................................................................................................................................ 59 1.6. Others......................................................................................................................................................................... 60 2. MetHods.........................................................................................................................................................61 2.1. Bacterial growth conditions............................................................................................................................. 61 2.2. Preparation of E. coli maxicells....................................................................................................................... 61 2.3. Complementation plate assay of E. coli VIP2 and PAT84.................................................................... 62 1 2.4. Production of FtsZ‐VM and FtsZ*‐VM in E. coli VIP2 and maxicells................................................62 2.5. BiFC in growing cells and in maxicells.........................................................................................................62 2.6. Overproduction of GFP‐MinD and MinE‐GFP ...........................................................................................63 2.7. P1 transduction......................................................................................................................................................63 2.8. Plasmids....................................................................................................................................................................63 2.9. Production of anti His‐MinE antibody .........................................................................................................66 2.10. Phase‐contrast and fluorescence microscopy .......................................................................................66 2.11. SDS‐PAGE and immunoblots.........................................................................................................................67 2.12. Protein structural data.....................................................................................................................................67 Results......................................................................................................................................................69 1. Maxicells: proof of principle ..................................................................................................................71 1.1. Screening of recA mutant strains for maxicell production .................................................................71 1.2. Loss of nucleoid in E. coli maxicells...............................................................................................................71
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