The sigma54 activator bypass problem in vivo and in vitro Jorrit Schäfer Division of Cell and Molecular Biology, Department of Life Sciences, Faculty of Natural Sciences. SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DECEMBER 2014 1 Abstract Tight regulation of gene expression is crucial for the survival of an organism, and allows certain genes to be switched on or off depending on the growth conditions and the state of differentiation. Transcription initiation is the most highly regulated step of gene expression, preventing wastage and being subject to the action of sophisticated signalling pathways. The RNA polymerases are further regulated through multiple different activators (stimulating transcription) and repressors (inhibiting transcription). Notably, promoter recognition specificity in bacteria is regulated by several dissociable sigma factors which can bind to the RNA polymerase core enzyme. Sigma54 (σ54) is the major alternative sigma factor in E. coli and historically known for its role in nitrogen metabolism. One unique property of σ54-dependent transcription is that it recognises -12 and -24 promoter elements rather than the traditional -10 and -35 sequences. Another distinguishing feature of σ54-dependent transcription initiation is that it absolutely requires the activity of a cognate activator and ATP hydrolysis, potentially giving tight control over gene expression and a wider dynamic range than with σ70-dependent transcription. Interestingly, this activator requirement has been shown to be bypassed in vitro with σ54 mutants where the interaction with the -12 promoter DNA element is disrupted. However, σ54-dependent transcription is not readily observed in vivo for the same mutants, suggesting further barriers exist in vivo inhibiting activator independence from occurring. The -12 promoter element has been shown to be somewhat expendable for σ54 promoter binding, and activator-independent σ54 transcription would obviate the need for additional proteins and ATP. This raises the question as to why σ54-dependent transcription has not evolved towards activator independence over time, by bypassing the interaction with the -12 promoter DNA. In this study, I show that σ54-dependent inhibition of transcription was detected for several genes with σ54 binding sites in their promoter region (ytfJ, chaC, patA, argT, mdfA ybhK, and acrD). Additionally, local transcriptional repression in the RNASeq data directly correlated with proximal σ54 binding sites, suggesting a novel role for σ54 as a transcriptional repressor. Strikingly, the -12 consensus motifs were more conserved in promoters linked to σ54- dependent transcriptional inhibition, underlining the importance of this promoter element in this newly discovered putative repressive function. However, extensive screens for genes inhibiting bypass transcription in vivo failed to identify any major repressive genes that keep activator independence in check in vivo. The 2 mechanisms maintaining activator bypass transcription at low levels in vivo still remain to be characterised. Table of Contents Abstract 2 Table of Contents 3 Declaration of originality 8 Copyright Declaration 8 Acknowledgements 8 List of Figures 9 List of Tables 11 Abbreviations 12 Chapter 1: Introduction 16 1.1 Gene expression and transcription in bacteria 16 1.2 Bacterial core RNAP 18 1.2.1 Multi-subunit RNAPs 18 1.2.2 Bacterial core RNAP structure 19 1.2.3 The alpha subunit 20 1.2.4 The beta subunit 21 1.2.5 The beta prime subunit 22 1.2.6 The omega subunit 23 1.3 The sigma (σ) factors 24 1.3.1 The sigma70 (σ70) family 26 1.3.2 The sigma70 (σ70) family subgroups 27 1.3.3 Sigma70-dependent (σ70) transcription initiation 28 1.4 The sigma54 (σ54) family 31 1.4.1 The E. coli sigma54 (σ54) 33 1.4.2 Bacterial enhancer binding proteins (bEBPs) 35 1.4.2.1 Bacterial enhancer binding proteins in E. coli 37 1.4.3 Sigma54-dependent (σ54) transcription initiation 38 1.4.3.1 Closed complex formation 38 1.4.3.2 Intermediate and open complex formation 39 1.4.4 Mutants of sigma54 (σ54) that bypass the activator requirement 41 54 1.4.4.1 Disruption of a leucine patch in σ RI 41 1.4.4.2 A two-step model for activation 41 3 1.4.4.3 The activator bypass phenotype and the -12 DNA interaction 42 1.4.4.4 The σ54 activator bypass mutant phenotype is not readily observed in vivo 42 1.4.5 Objectives 43 Chapter 2: Materials and Methods 44 2.1 Materials 44 2.1.1 Antibiotics 44 2.1.2 Media 44 2.1.3 Bacterial Strains 45 2.1.4 Kits 46 2.2 DNA Methods 47 2.2.1 Chromosomal DNA purification 47 2.2.2 Plasmid DNA purification 47 2.2.3 PCR 47 2.2.4 Agarose gel electrophoresis 48 2.2.5 Gel Extraction 48 2.2.6 DNA Sequencing 48 2.2.7 Restriction Digest 48 2.2.8 DNA Ligation 48 2.2.9 CaCl2 competent cells 49 2.2.10 Transformation (heat-shock) 49 2.3 In Vitro Methods 50 2.3.1 Beta-galactosidase assay 50 2.3.2 SDS-PAGE 50 2.3.3 Abortive transcription assay 51 2.3.4 Full length transcription assay 53 2.3.5 Inverse PCR 53 2.4 In Vivo Methods 54 2.4.1 P1vir phage transduction 54 2.4.2 GFP fluorescence and cell growth 54 2.4.3 RNA-Sequencing 54 Chapter 3: High temperature increases bypass transcription in vivo 56 3.1 Introduction and objectives 56 3.2 σ54 Activator Bypass Reporter Strain Construction 57 54 3.2.1 rpoN208::Tn10 abolishes σ -dependent transcription at the pspA promoter 58 3.2.2 Plasmid-borne wild type σ54 expression restores σ54 activity in rpoN208::Tn10 cells 60 54 3.2.3 Introducing Klebsiella pneumonia σ ΔR1 to construct a functioning SABRS in vivo 62 4 3.2.4 Confirmation of reporter strain genetic backgrounds of using colony PCR 63 3.2.5 The SABRS (with rpoNΔR1) does not cause SABT in vivo under standard conditions 65 3.2.6 pBAD18-rpoNR1 is expressed in the SABRS 66 3.3 Results 68 54 54 3.3.1 Wild type σ induces ΦPglnAp2-gfp expression, σ ΔR1 activator bypass mutant does not 69 54 54 3.3.2 Lower salt concentration does not induce ΦPglnAp2-gfp in vivo, with σ or σ ΔR1 71 54 54 3.3.3 L-glutamine concentration does not induce ΦPglnAp2-gfp in vivo, with σ or σ ΔR1 73 54 3.3.4 High temperature increases SABT at glnAp2 in vivo, and reduces wild type σ transcription 74 3.3.5 High temperature does not increases SABT at pspA promoter in vivo 76 3.3.6 Global bypass transcription in vitro using chromosomal DNA 78 3.4 Summary 81 Chapter 4: Genome-wide screens for genes regulating SABT in vivo 82 4.1 Introduction and objectives 82 4.1.1 Recap and objectives 82 4.1.2 Introduction to KEIO collection 83 4.1.3 Introduction to generalised P1vir phage transduction 84 4.1.4 Identifying KEIO mutants using inverse PCR 86 4.2 Results and Discussion 87 4.2.1 KEIO deletions and ΦPpspA-LacZ activity in the pspA-SABRS 88 4.2.2 Co-transduction of wild type chromosomal rpoN 89 4.2.3 Screening KEIO library mutants for SABT at increased arabinose concentration 90 4.2.4 Screening KEIO mutants for ΦPpspA-lacZ activity at increased temperature 92 4.2.5 Small peptide/RNA deletion library and ΦPpspA-LacZ activity in the SABRS 93 4.2.6 Salmonella Typhimurium LT2 overexpression clones and SABT 94 4.2.7 Selection of activator bypass mutants on minimal lactose media 96 4.3 Discussion 99 4.3.1 Function of genes involved in putative repression of SABT in vivo 99 4.3.2 ΔasnA as a candidate for putative bypass transcription 100 4.3.3 Low levels of σ70-dependent transcription of the pspA promoter in E. coli 101 4.3.4 ΔyhbX and ΔnanT as candidates for putative bypass transcription 103 4.3.5 ΔgreA as a candidate for putative bypass transcription 103 4.3.6 ΔhldE as a candidate for putative bypass transcription 104 4.3.7 ΔttdR as a candidate for inhibiting putative bypass transcription 106 4.4 Summary 107 5 54 Chapter 5: Novel role for σ as a transcriptional repressor 108 5.1 Introduction and objectives 108 5.1.1 Recap and objectives 108 5.1.2 Introduction to RNA Sequencing 109 5.1.3 rRNA depletion and RNA Sequencing in E. coli 110 5.1.4 First strand priming and synthesis 111 5.1.5 Second strand priming and synthesis 113 5.1.6 5’ end processing and sequencing adapters 114 5.1.7 Illumina Next Generation Sequencing 114 5.1.8 A typical RNA Sequencing experiment for E. coli 116 5.2 Results 117 5.2.1 SABT is detectable at the glnAp2 promoter 118 5.2.2 Gene repression in the SABRS 120 5.2.3 Gene upregulation in the SABRS 123 5.2.4 Gene repression in the WTRS 126 5.2.5 Gene upregulation in the WTRS 128 5.2.6 Comparison of RNASeq data and σ54 chIP binding study 130 5.2.7 Putative SABT in SABRS in genes with σ54 binding sites. 131 5.2.8 Upregulation of genes with σ54 binding sites in WTRS 132 5.2.9 Repression of genes with σ54 binding sites in WTRS 133 5.2.10 Repression in WTRS of genes with σ54 binding sites upstream of gene 134 5.2.11 Putative repressive σ54 binding sites and local repression of transcription 135 5.3 Discussion 138 54 54 5.3.1 Gene upregulation by wild type σ and activator bypass σ ΔR1 138 5.3.2 Putative repressive σ54 binding motifs around transcription start sites 139 5.3.3 Alignment of σ54 consensus sequences involved in activation, SABT and repression 142 5.3.4 σ54 and its potential role in the transcription of cold shock genes 145 5.4 Summary 147 Chapter 6: Transcription of cold shock genes and SABT in vitro 148 6.1 Introduction and objectives 148 6.1.1 Recap and objectives 148 6.1.2 Abortive initiation assays 149 6.2 Results 150 6 6.2.1 Abortive initiation assay with nifH promoter DNA 150 6.2.2 Abortive initiation assay at prpB promoter 152 6.2.3 Abortive initiation assay at hypA promoter 153 6.2.4 Abortive initiation assay at yahE promoter 155 6.2.5 Abortive initiation assay at pspG promoter 157 6.2.6 Abortive initiation assay at cspA promoter 159 6.2.7 Abortive initiation assay at cspH promoter 161 6.3 Summary 163 Conclusion 165 Future Work 167 Bibliography 168 Appendix A (Primers) 191 Appendix B (RNASeq) 192 54 Appendix C (σ ChIP study) 197 7 Declaration of originality I, Jorrit Schäfer declare that I am the sole author of the written work in this thesis and that all the results are original.