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Regulation of Phenotypic Switching and Heterogeneity in Photorhabdus Luminescens Cell Populations

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Regulation of Phenotypic Switching and Heterogeneity in Photorhabdus Luminescens Cell Populations Regulation of phenotypic switching and heterogeneity in Photorhabdus luminescens cell populations Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Angela Katharina Langer geb. Glaeser am 17.11.2016 1. Gutachter: PD Dr. Ralf Heermann, LMU München 2. Gutachter: Prof. Dr. Marc Bramkamp, LMU München Datum der Abgabe: 17.11.2016 Datum der mündlichen Prüfung: 12.12.2016 II Eidesstattliche Erklärung Ich versichere hiermit an Eides statt, dass die vorgelegte Dissertation von mir selbstständig und ohne unerlaubte Hilfe angefertigt wurde. Des Weiteren erkläre ich, dass ich nicht anderweitig ohne Erfolg versucht habe, eine Dissertation einzureichen oder mich der Doktorprüfung zu unterziehen. Die folgende Dissertation liegt weder ganz, noch in wesentlichen Teilen einer anderen Prüfungskommission vor. München, den 17.11.2016 Angela Langer Statutory Declaration I declare that I have authored this thesis independently, that I have not used other than the declared sources/references. As well I declare that I have not submitted a dissertation without success and not passed the oral exam. The present dissertation (neither the entire dissertation nor parts) has not been presented to another examination board. Munich, 17.11.2016 Angela Langer III Contents Eidesstattliche Erklärung ...................................................................................................................... III Statutory Declaration............................................................................................................................ III Contents ...............................................................................................................................................IV Nomenclature .......................................................................................................................................VI Abbreviations ..................................................................................................................................... VII Publications and Manuscripts presented in this thesis ..........................................................................IX Contributions to publications and manuscripts presented in this thesis ................................................. X Summary ............................................................................................................................................ XII Zusammenfassung ............................................................................................................................. XIV 1 Introduction .................................................................................................................................... 1 1.1 The genus Photorhabdus ........................................................................................................ 1 1.1.1 Life cycle of Photorhabdus luminescens ........................................................................ 1 1.1.2 Quorum sensing in Photorhabdus species ...................................................................... 3 1.1.3 Secondary metabolism in Photorhabdus species ............................................................ 6 1.2 Phenotypic heterogeneity ....................................................................................................... 7 1.2.1 Different types of phenotypic heterogeneity ................................................................... 8 1.2.2 Phenotypic heterogeneity in P. luminescens ................................................................. 10 1.3 LysR-type transcriptional regulator (LTTRs) ....................................................................... 12 1.3.1 Structure and function of LTTRs .................................................................................. 13 1.3.2 HexA in Photorhabdus and other organisms ................................................................ 14 1.4 Scope of this thesis ............................................................................................................... 15 1.5 References of introduction .................................................................................................... 16 2 A novel tool for stable genomic reporter gene integration to analyze heterogeneity in Photorhabdus luminescens at the single-cell level ............................................................................... 24 3 Heterogeneous regulation of bacterial natural biosynthesis via a novel transcription factor ......... 26 4 HexA is a versatile regulator involved in the control of phenotypic heterogeneity of Photorhabdus luminescens ................................................................................................................... 28 5 Social biological relevance of the two phenotypic cell forms for the population of Photorhabdus luminescens ................................................................................................................... 63 5.1 Introduction .......................................................................................................................... 63 5.2 Material and Methods ........................................................................................................... 64 5.2.1 Materials ....................................................................................................................... 64 5.2.2 Bacterial strains and growth conditions ........................................................................ 65 5.2.3 Plasmid generation ....................................................................................................... 66 5.2.4 Competent cells and transformations ............................................................................ 66 5.2.5 Integration of reporter genes into the P. luminescens genome ...................................... 66 5.2.6 Growth analysis of 1° and 2° cells ................................................................................ 67 IV 5.3 Results .................................................................................................................................. 67 5.3.1 2° cells outcompete 1° cells in the stationary phase ...................................................... 67 5.3.2 Influence of stress conditions on the growth behavior in a growth competition assay of 1° and 2° cells............................................................................................................................... 70 5.4 Discussion ............................................................................................................................ 71 5.5 References of Chapter 5 ....................................................................................................... 73 6 Concluding Discussion ................................................................................................................. 76 6.1 Heterogeneity in the regulation of secondary metabolism .................................................... 76 6.2 The regulatory network of phenotypic switching ................................................................. 80 6.2.1 Regulation of hexA expression...................................................................................... 81 6.2.2 Regulation of primary-specific features via HexA ........................................................ 82 6.2.3 Potential role of the two phenotypic forms for a P. luminescens population................. 84 6.3 Outlook ................................................................................................................................. 87 6.4 References of Concluding Discussion .................................................................................. 89 Supplemental Material Chapter 2 ......................................................................................................... 94 Supplemental Material Chapter 3 ......................................................................................................... 95 Supplemental Material Chapter 4 ......................................................................................................... 96 Acknowledgments .............................................................................................................................. 109 V Nomenclature Gene products are numbered in a way that the first methionine of the wild-type protein is designated “1” in the amino acid sequence (if present: independently of the N-terminal affinity tag). N-terminal and C-terminal affinity tags are marked in genes and proteins corresponding to their position (e.g. 6His-HexA or HexA-6His). Deletions of genes are marked by “Δ”. Unless otherwise noted, nucleotide positions indicate the distance from the transcriptional start site (+1). The two phenotypic forms of Photorhabdus luminescens TT01 are called primary and secondary cells and can also be presented as TT01-1° and TT01-2° or 1° and 2°. VI Abbreviations aa amino acid AHL acyl homoserine lactone AQ anthraquinone ATP adenosine-5’-triphosphate AU arbitrary unit bp base pairs cfu colony forming units DNA deoxyribonucleic acid DNase deoxyribonuclease DTT 1,4-Dithiothreitol IJ infective juvenile JHEH juvenile hormone epoxide hydrolase EMSA electrophoretic mobility shift assay EYFP enhanced yellow fluorescent protein n-His tag affinity tag composed of n histidine residues HexA hyperproduction of exoenzymes
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