Investigating the Relationship Between Quorum Sensing, Motility, and the Type 3 Secretion System Of
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Investigating the relationship between Quorum Sensing, Motility, and the Type 3 Secretion System of Yersinia pseudotuberculosis Robert J. Goldstone Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy September 2011 i Declaration Unless otherwise acknowledged, the work presented in this thesis is entirely my own. No part has been submitted for another degree in the University of Nottingham or any other institute of learning. Robert Goldstone September, 2011 ii Abstract Over the course of the last two decades, research into the role of quorum sensing (QS) in regulating diverse bacterial behaviours has exploded, and around twelve years ago, a QS network was identified in the enteropathogenic bacterium Yersinia pseudotuberculosis, which was shown to control motility and cellular clumping. This thesis seeks to expand this regulatory relationship and explore the causes and consequences of the link between QS and motility, which affects pleiotropic processes including the type 3 secretion system (T3SS) and biofilm formation. Indeed, the clumping phenotype first explored by Atkinson et al. (1999), is linked to QS-dependent regulation of the T3SS, since the deletion of several QS genes results in liquid culture biofilm (LCB) formation. This is concomitant with T3S protein secretion into culture supernatant, which occurs under normally non-inducing conditions, while deleting the T3SS structural component yscJ prevents secretion and LCB formation. De-repression of the T3SS and the development of LCBs also occurs following mutation of the flagella regulators flhDC and fliA, revealing that QS and the flagella system co-regulate LCBs. However, interestingly it was found that LCB formation and secretion also occurs following mutation of the flagella structural gene flhA. The ∆flhA mutant represents a flagella-minus strain, in which the underlying regulatory circuit mediated by FlhDC and FliA is intact, suggesting that an element of the flagella structure that depends on FlhA activity acts as a check-point governing expression of the T3SS. Both QS and the flagella system positively regulate biofilm formation by Y. pseudotuberculosis on the surface of the nematode worm, Caenorhabditis elegans. Surprisingly, the up-regulated T3SS was found to be responsible for mediating down- regulation of biofilm formation by Y. pseudotuberculosis QS mutants, since subsequent deletion of yscJ could restore biofilms to wild-type levels. This suggested that a component of the injectisome was capable of influencing cellular processes in addition to its role in secretion. In light of the link regulatory link between flagella and T3S, this raised the possibility that the injectisome could play a role in the reciprocal regulation of motility. Since the genetic regulatory network underpinning expression of the T3SS is intact in the ∆yscJ mutant, like the ∆flhA mutant for flagella, the ∆yscJ mutant can reveal the role of the injectisome structure in modulating gene expression. By phenotypic observation, it was determined that the ∆yscJ mutant displayed aberrant flagella mediated motility, swimming vigorously under conditions in which the wild-type did not, and, similar to the over-production of Yop proteins in the ∆flhA mutant, the ∆yscJ mutant over-produces flagellin. This suggests that a component of the T3SS injectisome acts as a checkpoint to regulate motility, which appears to be at the level of transcription, since the ∆yscJ mutant displays up-regulation of the flagella regulators flhDC and fliA. Indeed, the relationship between T3S and motility appears to require a direct influence on QS, since subsequent mutation of ypsI and ytbI abolishes ∆yscJ-dependent hyper-motility, the ∆yscJ mutant displays altered expression of the QS system genes. Furthermore, for the emerging transcriptional relationship between these systems, the flagella and QS mutants which are up-regulated for the production of Yop proteins also over-express the virulence regulator virF, completing the transcriptional regulatory circuit which appears to be crucial for the regulation of lifestyle choices by Y. pseudotuberculosis. iii Publications arising from this work Sections of this thesis have appeared in the following publications: Atkinson S, Goldstone RJ, Joshua GWP, Chang C-Y, Patrick HL, Cámara M, Wren BW, Williams P, (2011) Biofilm Development on Caenorhabditis elegans by Yersinia Is Facilitated by Quorum Sensing-Dependent Repression of Type III Secretion. PLoS Pathogens 7: e1001250. Goldstone RJ, Popat R, Fletcher MP, Crusz SA, Diggle SP, (2012) Quorum sensing and social interactions in microbial biofilms. In Microbial Biofilms: current research, methods and applications (Ed. Lear & Lewis). Caister Academic Press. iv Acknowledgements First and foremost I would like to thank Professor Paul Williams for providing me with the opportunity to undertake this work in his laboratory, for the help and guidance he has provided me with throughout, and for the freedom he has granted me to pursue my own interests in the lab. I would also like to thank Dr. Steve Atkinson for all his diligent support and supervision, for his help and all the ideas we bounced between one another which made this PhD such an exciting and interesting project. I also thank Professor Miguel Cámara for his ideas, help and support- I could not imagine having three better supervisors. A great number of people in the CBS and Nottingham have made my PhD an unforgettable experience; to list them all would take longer than it will take you, dear reader, to read this thesis. But a particular mention must go to three friends who came through all this with me: Dave, Roman and Avika. We started together and we finished together, I’m so glad we came all this way together. I would also like to thank Fiona for putting up with me through all of this, and my parents, for their support when I needed it most. This is for all of you. I also gratefully acknowledge Mark Davis and Mologic Ltd., alongside the BBSRC for their contribution towards funding this work. Companies such as theirs, which take chances in funding research such as this, deserve a great deal of success in their business. Robert Goldstone v Table of contents Title i Declaration ii Abstract iii Publications arising from this work iv Acknowledgements v Table of Contents vi Figures & Tables xv Abbreviations xix Chapter 1| Introduction 1.1 Bacterial Communication and Quorum Sensing 2 1.1.1 AHL signalling and QS 3 1.1.2 LuxI family proteins and signal generation 5 1.1.3 LuxR-family proteins and signal transduction 7 1.1.4 AHL signalling in virulence 10 1.2 Yersinia pseudotuberculosis 11 1.2.1 Diseases caused by the yersiniae 12 1.2.2 The environmental control of virulence 14 1.2.3 The Yersinia virulence plasmid 15 1.2.4 The Yop-Ysc type 3 secretion system 16 1.2.4.1 The structure of the Yop-Ysc T3SS 18 vi 1.2.4.2 Sorting proteins for the T3SS 20 1.2.4.3 Chaperones facilitate the T3SS 21 1.2.4.4 The Yop effectors 22 1.2.5 Regulation of the Yop-Ysc T3SS 25 1.2.5.1 The genetic control of the Yop-Ysc T3SS 26 1.2.6 Other Yersinia virulence factors 28 1.2.6.1 The Pgm locus 28 1.2.6.2 The adhesins- Inv, Ail, the pH 6 antigen and YadA 29 1.2.7 QS in the yersiniae 32 1.3 Biofilms- On Surfaces and In Liquid Culture 34 1.3.1 Architecture of biofilms 35 1.3.2 Function of biofilms 37 1.3.2 Biofilm development 39 1.3.2 Yersinia biofilms 41 1.3.2 QS in biofilms 42 1.4 Flagella Mediated Motility 45 1.4.1 The structure and function of the flagellum 45 1.4.2 QS and the genetic regulation of flagella 49 1.5 Aims 50 vii Chapter 2| Materials and Method 2.1 Growth Conditions 53 2.1.1 Growth media 53 2.1.2 Growth conditions 55 2.1.2 Bacterial strains 55 2.2 Genetic Manipulation 60 2.2.1 DNA 60 2.2.2 Plasmids used in this study 60 2.2.3 Restriction enzymes 62 2.2.4 Separation of DNA by agarose gel electrophoresis 62 2.2.5 DNA ligation 63 2.2.6 Polymerase chain reaction conditions 63 2.2.6.1 Synthesis of oligonucleotide primers 63 2.2.6.2 PCR amplification 65 2.2.7 Introducing DNA into bacterial cells 65 2.2.7.1 Preparation of electro-competent cells 65 2.2.7.2 Electroporation 66 2.2.7.3 Conjugation 66 2.2.8 Construction of the virF promoter fusion 67 2.2.9 Mutagenesis of yscJ 67 2.2.10 Transposon mutagenesis 68 2.2.11 Sequencing of DNA 69 viii 2.3 Extraction and Analysis of Proteins 70 2.3.1 Preparation of supernatant proteins 70 2.3.2 Purification of flagella 70 2.3.3 TCA precipitation 71 2.3.4 SDS-PAGE 71 2.3.5 Protein profiling and sequencing 71 2.4 Phenotypic Assays 72 2.4.1 Liquid culture biofilm assays 72 2.4.2 Congo red binding in liquid culture 74 2.4.3 Measuring biofilms on the surface of C. elegans 74 2.4.3.1 Biofilms grown in the agar method 74 2.4.3.2 Biofilms grown in the compost method 75 2.4.4 Determination of luminescence and optical density 76 2.5 Microscopy 77 ix Chapter 3| QS and liquid culture biofilms 3.1 Introduction 79 3.1.1 Cellular physiology and genetics of LCBs 80 3.1.2 LCBs in the yersiniae 83 3.1.3 Chapter 3 aims 86 3.2 Results 88 3.2.1 QS and LCBs 88 3.2.1.1 QS controls LCB formation 88 3.2.1.2 YpsI and YtbI are necessary for LCBs 90 3.2.2 Biofilm matrix components in LCBs 92 3.2.2.1 Extracellular DNA and polysaccharides in LCBs 92 3.2.2.2 DNA is an important structural component of LCBs 97 3.2.3 Supernatant factors influence LCB formation 99 3.2.3.1 Secretion is involved in LCB formation 99 3.2.3.2 Boiling ∆ypsI ∆ytbI mutant supernatant prevents LCB formation 101 3.2.3.3 Excluding macromolecules from supernatant prevents LCB 102