SINGLE-MOLECULE STUDIES ON THE ROLE OF NUCLEOID-ASSOCIATED PROTEINS IN BACTERIAL CHROMATIN RICKSEN SURYA WINARDHI NATIONAL UNIVERSITY OF SINGAPORE 2014 SINGLE-MOLECULE STUDIES ON THE ROLE OF NUCLEOID-ASSOCIATED PROTEINS IN BACTERIAL CHROMATIN RICKSEN SURYA WINARDHI B.Sc.(Hons.), Nanyang Technological University A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 Declaration I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Ricksen Surya Winardhi 31 July 2014 Acknowledgements The research works done in this thesis are supported by NUS Graduate School for Integrative Sciences and Engineering at the National University of Singapore. My journey to complete this thesis would not have been possible without the help and support from many people: I would like to thank my supervisor, Assoc. Prof. Yan Jie, for being the best Ph.D. supervisor. I felt lucky and privileged to my research works under his direction and supervision, as he led me patiently for years towards the completion of this thesis. I learnt many lessons from him that I will never forget in my life: his love and enthusiasm for science, his desire for each of his students to succeed in whatever field he/she does, his highly positive attitude towards others’ critics which give room for improvements, his attitude of not blaming others in unwelcome situations, his remarkable perseverance and never-give-up attitude, as well as many others. My gratitude to my thesis advisory committee: Assoc. Prof. Wang Zhisong as the committee chair, Assoc. Prof. Thorsten Wohland, and Asst. Prof. Cynthia He, for their advices and direction for this thesis. iii Acknowledgements iv My colleagues in single-molecule biophysics laboratory: Lim Ci Ji for the stimulating discussions, advices, and constructive feedbacks; Fu Wenbo and Li Yanan for their contributions during the initial stage of MvaT project; Ranjit Gulvady for his help in the Ler project; as well as for Chen Hu, Fu Hongxia, Liu Yingjie, Saranya Chandrasekar, Le Shimin, Yao Mingxi, Yuan Xin, Artem Yefremov, Lee Sin Yi, Li You, Zhang Xinghua, Zhao Xiaodan, Qu Yuanyuan, Lin Jie, Xu Yue, Low Yee Teck, Zhao Wenwen, and many others. Importantly, I want to thank my parents, family, and friends, who have supported me. My fianc`ee Marissa Iskandar for her continuous support and prayer. Their presence, support, and encouragement are substantial and immensely meaningful for me. Ricksen Surya Winardhi July 2014 Contents Declaration ii Acknowledgements iii Summary viii List of Figures Introduction 1.1 Background of the Study . . . . . . . . . . . . . . 1.2 Literature Review . . . . . . . . . . . . . . . . . . 1.2.1 Structure and Genetics of Bacteria . . . . 1.2.2 Bacterial DNA Organisation . . . . . . . . 1.2.3 Regulation of Gene Expression in Bacteria 1.3 Objective of the Study . . . . . . . . . . . . . . . 1.4 Thesis Outline . . . . . . . . . . . . . . . . . . . . x . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental Techniques 2.1 Magnetic Tweezers . . . . . . . . . . . . . . . . . . . . . . 2.1.1 Experimental protocol . . . . . . . . . . . . . . . . 2.1.2 Force calibration . . . . . . . . . . . . . . . . . . . 2.1.3 Worm-like chain polymer under force . . . . . . . . 2.1.4 E↵ects of protein binding on DNA micromechanics 2.2 Atomic Force Microscopy . . . . . . . . . . . . . . . . . . . 2.2.1 Mica surface modification . . . . . . . . . . . . . . 2.2.2 The instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 10 11 . . . . . . . . 14 15 16 20 20 21 23 25 27 The formation of nucleoprotein filaments and higher order oligomerization are required for MvaT silencing activity in Pseudomonas aeruginosa 28 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 v Contents 3.2 3.3 3.4 Materials and Methods . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1 MvaT simultaneously sti↵ens and folds DNA in single DNA stretching experiments . . . . . . . . . . . . . . 3.3.2 MvaT binds cooperatively to DNA . . . . . . . . . . 3.3.3 MvaT forms nucleoprotein filaments and compact DNA structures in single-molecule imaging experiments . . 3.3.4 E↵ects of variation in environmental factors to MvaT nucleoprotein filaments and MvaT-induced DNA folding . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.5 Functionally defective MvaT mutants cannot form nucleoprotein filaments . . . . . . . . . . . . . . . . . 3.3.6 MvaT nucleoprotein filaments restrict DNA accessibility . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1 The organisation modes of MvaT to DNA . . . . . . 3.4.2 Implications of MvaT nucleoprotein filament formation on gene silencing . . . . . . . . . . . . . . . . . . 3.4.3 Implications of MvaT-induced DNA folding on chromosomal DNA packaging . . . . . . . . . . . . . . . . Single-molecule study on Histone-like Nucleoid-structuring Protein (H-NS) Paralogue in Pseudomonas aeruginosa: MvaU Bears DNA Organisation Mode Similarities to MvaT 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Materials & Methods . . . . . . . . . . . . . . . . . . . . . . 4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 MvaU can sti↵en and fold DNA in single molecule stretching experiments . . . . . . . . . . . . . . . . . 4.3.2 MvaU organises DNA into higher-order rod-like structures and compact DNA structures . . . . . . . . . . 4.3.3 Variation in environmental factors can modulate MvaUinduced DNA folding . . . . . . . . . . . . . . . . . . 4.3.4 MvaU nucleoprotein filaments can protect DNA from DNase1 cleavage . . . . . . . . . . . . . . . . . . . . 4.3.5 Mixture of MvaT and MvaU sti↵en and fold DNA in single-molecule stretching experiments . . . . . . . . 4.4 Discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1 The organisation mode of MvaU to DNA . . . . . . . 4.4.2 The implication of MvaU binding on its functional role 4.4.3 Comparison between MvaT’s and MvaU’s DNA organisation mode and their roles in vivo . . . . . . . . Ler can antagonize H-NS nucleoprotein filaments through vi 31 33 33 35 40 46 51 55 59 59 61 63 65 66 68 70 70 72 74 78 80 82 82 83 84 Contents vii non-cooperative DNA binding 86 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 5.2 Materials & Methods . . . . . . . . . . . . . . . . . . . . . . 89 5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 5.3.1 Ler binds to extended DNA and increases DNA bending rigidity . . . . . . . . . . . . . . . . . . . . . . . 90 5.3.2 Ler binds to extended DNA through non-cooperative binding process . . . . . . . . . . . . . . . . . . . . . 94 5.3.3 Ler can fold DNA through association of Ler-bound DNA with naked DNA . . . . . . . . . . . . . . . . . 97 5.3.4 Ler forms compact DNA structures and extended DNA structures in single-molecule imaging experiments . . . . . . . . . . . . . . . . . . . . . . . . . . 101 5.3.5 Ler responses to environmental factors . . . . . . . . 105 5.3.6 Ler replaces prebound H-NS from DNA . . . . . . . . 112 5.3.7 E↵ect of KCl and MgCl2 concentration on H-NS and Ler binding to DNA . . . . . . . . . . . . . . . . . . 115 5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 5.4.1 The organisation modes of Ler to DNA . . . . . . . . 119 5.4.2 Implications of Ler responses to environmental factors 123 5.4.3 Implications on Ler mediated anti-silencing activity . 124 Conclusion 126 6.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 6.1.1 The formation of nucleoprotein filaments and higher order oligomerization are required for MvaT silencing activity in Pseudomonas aeruginosa . . . . . . . . . . 126 6.1.2 Single-molecule study on Histone-like Nucleoid-structuring Protein (H-NS) Paralogue in Pseudomonas aeruginosa: MvaU Bears DNA Organisation Mode Similarities to MvaT . . . . . . . . . . . . . . . . . . . . . 127 6.1.3 Ler can antagonize H-NS nucleoprotein filaments through non-cooperative DNA binding . . . . . . . . . . . . . 128 6.2 Relevance and Outlook . . . . . . . . . . . . . . . . . . . . . 129 Publications & Scientific Meetings 132 Bibliography 132 Summary Bacterial chromatin contains the genetic code of bacteria, assembled by many factors into a compact structure called nucleoid. The primary focus of this thesis is on the role of nucleoid-associated proteins in shaping the bacterial nucleoid and regulating the gene expression. Despite wealth of knowledge on the function of these proteins obtained by biochemical studies, little is known regarding their molecular mechanism. This gap of knowledge can be bridged by biophysical techniques, which are capable to probe the DNA binding properties of these important proteins at the singlemolecule level. Single-molecule manipulation using magnetic tweezers and single-molecule imaging using atomic force microscopy were utilized in the works leading to this thesis to gain insights into the molecular mechanisms of gene regulation and DNA packaging in bacteria. Each year, bacterial pathogens cause infections leading to innumerable illnesses, hospitalizations, and deaths. The virulence gene expression of these pathogens is often regulated by these gene silencing and anti-silencing proteins. In cystic fibrosis (CF) patients, for example, Pseudomonas aeruginosa infects and persists in the lung as colonies encased in a matrix called viii Summary ix biofilm, which can increase their resistance towards antibiotics. These virulence gene expression and biofilm formation are regulated by important gene silencing proteins MvaT and MvaU. In pathogenic E. coli, the interplay between H-NS and Ler protein is crucial in regulating the pathogenicity island containing the genes responsible for causing severe infantile diarrhoea, haemorrhagic colitis, hemolytic-uremic syndrome, etc. In this thesis, I present my work on global gene silencing protein in Pseudomonas aeruginosa, MvaT, which is a member of H-NS-family proteins. I show that MvaT can form rigid nucleoprotein filaments, while its functionally defective and higher-order oligomerization defective mutants cannot form such filaments. 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[...]... force-extension curves of naked DNA as our control experiment By changing the bu↵er to those containing DNA-binding protein of our interest, we can study the DNA binding properties of the protein by its impact on DNA elastic response (Fig 2.3) Theoretical predictions have shown that binding of DNA-distorting proteins can change the force-extension curves of DNA, hence giving us information on the binding mechanism... reorganisation of the nucleoid structure Furthermore, an analysis on the 12 major NAPs revealed changes in the composition of these proteins depending on the bacterial growth phase [26] Since these proteins have di↵erent modes in organising DNA, the nucleoid structure is dynamically modulated to di↵erent compacted states due to di↵erent level of expression of the major architectural proteins 1.2.3 Regulation of. .. that the protein-DNA interaction energy is in the range of kB T , the force generated on the nucleoid structure can be up to few pN 2 1.2 Literature Review This lack of understanding on the DNA binding mechanisms of important bacterial proteins, together with the neglect of force, become the roadblocks to better understand their underlying role and function Unravelling their mechanisms of action can... transcription? Second, the potential role of these proteins in chromosomal DNA packaging will be explored 10 1.4 Thesis Outline 1.4 11 Thesis Outline This thesis describes the scientific work done during my PhD candidature at the National University of Singapore The background and motivation of the research work are presented in this chapter Chapter two describes the main experimental techniques used in the. .. complexes Combined together, these two major single molecule methods of manipulation and imaging allow us 14 2.1 Magnetic Tweezers to ”feel” and ”see” the molecules of our interest Each of the single- molecule techniques have their own strengths and limitations Here we describe the main techniques used in our study of DNA and protein interactions: magnetic tweezers for single- molecule manipulation, and atomic... used in our experiments The upper panel shows the screenshot taken during one of the measurements Gray shaded area on the left part of the image is the thin polished cover slip, and the dark grey line is the cover slip edge This edge is taken as the reference point from the centroid of the magnetic bead to determine the extension of the stretched DNA Note that when the extension of the DNA is ⇠ 3 µm, the. .. that can antagonize gene silencing are key to better understand the molecular mechanism of gene regulation 1.3 Objective of the Study At the end of the study, we aim to better understand the DNA organisation mode of several bacterial nucleoid- associated proteins at single molecule level, which may provide us with invaluable information on how these important bacterial proteins achieve their in vivo regulatory... wealth of information on the binding properties of these and many other proteins 2.2 Atomic Force Microscopy The atomic force microscope uses the interaction of a sharp tip with sample surface to obtain the height profile or topography of the surface The size of the tip end, which are normally a few nanometers in radius, will determine the lateral resolution of the experiment This tip is mounted on a... of bacteria These processes are aided by many important proteins Biochemical studies have identified and discovered the function of these proteins and their regulatory pathways In addition to finding the functions of these proteins, it is equally important to understand how they perform their function, i.e their molecular mechanisms, which are mainly due to their DNA binding activity Moreover, many in. .. image of the bead is near the edge of our observation area and may interfere with the measurement Therefore, we didn’t let the extension of the DNA to go lower than this limit The attachment of DNA to the cover slip edge and magnetic bead is done through streptavidin-biotin ligand interactions The position of the pair of permanent magnet is adjusted to control the magnitude of force applied on the paramagnetic . SINGLE- MOLECULE STUDIES ON THE ROLE OF NUCLEOID- ASSOCIATED PROTEINS IN BACTERIAL CHROMATIN RICKSEN SURYA WINARDH I NATIONAL UNIVERSITY OF SINGAPORE 2014 SINGLE- MOLECULE STUDIES ON THE ROLE OF NUCLEOID- ASSOCIATED. of this thesis is on t h e role of nucleoid- associated proteins in sh ap i ng the bacterial nucleoid and regulating the gene expression. Despite wealth of knowledge on the function of these proteins. mechanisms. The novel findings in this thesis provide valuable insights and extend our understanding on the role of nucleoid- associated proteins in bacterial chromatin. Ricksen Surya Winardhi July