Development and Application of Synthetic Riboswitches as Tools to Study Bacterial Pathogenesis Open Access

Reynoso, Colleen Knight (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/b5644s63k?locale=en
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Abstract



In the laboratory, we require tools that allow conditional regulation of gene
expression to facilitate the study of genes with unknown or little understood function.
The ideal tool would provide translation of external stimuli to an internal alteration in
gene expression for any gene to be studied. Furthermore, these tools should be
transferrable between different species of bacteria to allow manipulation of expression in
less understood prokaryotic species. Nature has the advantage of billions of years of
evolution to determine efficient mechanisms for gene regulation. We have looked to
nature for inspiration and adapted natural systems to serve alternative purposes in the
laboratory. In this thesis we describe the development of synthetic riboswitches as
orthogonal tools to conditionally express bacterial genes of interest. We will explore the
concept of isolating functional riboswitches from a genetically tractable species of
bacterial and transporting these tools with limited alteration to less genetically tractable
species. Each chapter focuses on a different bacterial species with relevance to different
areas of microbiology and biotechnology and presents what we have learned regarding
the portability of synthetic riboswitches.

Table of Contents

Contents
Chapter 1: Introduction…………………………………………………………………1
1.1 Genetic Control in Response to Environmental Stimuli ………………………….......1
1.2 Sensing the Environment with RNA-Small Molecule Interactions: Evidence of the
RNA World ……………………………………………………………………………….3
1.3 Engineered Gene Regulation …………………………………………………………5
1.4 References ……………………………………………………………………...……14
Chapter 2: Observing Assembly of a Magnetosome protein in Magnetospirillum
magneticum Using Synthetic Riboswitches……………………………………………18
2.1 Introduction…...……………………………………………………………………...18
2.2 Results and Discussion.……………………………………………………………...23
2.3 Conclusion...….……………………………………………………………………...24
2.4 Experimental.………………………………………………………………………...25
2.5 References….………………………………………………………………………...28
Chapter 3: Screening for Synthetic Riboswitches in Acinetobacter baylyi …………32
3.1 Introduction…...……………………………………………………………………...32
3.2 Results and Discussion……….……………………………………………………...34
3.3 Conclusion…………………………………………………………………………...44
3.4 Experimental…………………………………………………………………………45
3.5 References……………………………………………………………………………49
Chapter 4: Developing Synthetic Riboswitches for use in Streptococcus
pyogenes…………………………………………………………………………………52
4.1 Introduction…...……………………………………………………………………...52
4.2 Results and Discussion……….……………………………………………………...56
4.3 Conclusion…………………………………………………………………………...69
4.4 Experimental…………………………………………………………………………70
4.5 References……………………………………………………………………………74
Chapter 5: Intracellular Study of Genes Involved in Francisella Pathogenesis……79
5.1 Introduction…...……………………………………………………………………...79
5.2 Results and Discussion……….……………………………………………………...81
5.3 Conclusion……………………………………………………………………..….…93
5.4 Experimental…………………………………………………………………………96
5.5 References…………………………………………………………………………..103
Chapter 6: Conclusions
6.1 Summary and Conclusions…………………………………………………………107
6.2 References…………………………………………………………………………..110
List of Figures
Figure 1.1 - Mechanisms of Natural Prokaryotic Riboswitches………………………….4
Figure 1.2 - SELEX Aptamer Selection Procedure………………………………………8
Figure 2.1 - Mechanism of translational theophylline-sensitive synthetic riboswitch…..19
Figure 2.2 - Magnetosomes within M. magneticum……………………………………..21
Figure 2.3 - Measure of reporter gene activity induced by riboswitch F in E. coli……..22
Figure 2.4 - Riboswitch-mediated induction of MamK-GFP…………………………...24
Figure 2.5 - Alignment of riboswitch F RBS with 16S rRNA of M. magneticum……..25
Figure 3.1 - Diagram of the location of the N8 library in the 5'-UTR………………….34
Figure 3.2 - Effect of ACC mutation on the linker region of the theophylline-inducible
riboswitch…………………………………………………….…………………………..35
Figure 3.3 - Schematic of the blue-white screen………………………………………...36
Figure 3.4 - Hydrolysis of PNPG by β-glucuronidase…………………………………..37
Figure 3.5 - β-glucuronidase activities for riboswitches identified from the A. baylyi
library screens……………………………………………………………………………39
Figure 3.6 - Proposed mechanism of A. baylyi riboswitch isolated from library
screens……………………………………………………………………………………40
Figure 3.7 - Comparison of "switch pack" riboswitches in A. baylyi and E. coli…….…41
Figure 3.8 - Comparison of 16S rRNA of A. baylyi and E. coli in context of riboswitch
RBS strength………………………………………………………………………….….43
Figure 4.1 - Streptococcus pyogenes morphology………………………………………53
Figure 4.2 - Theophylline toxicity in S. pyogenes strain JRS1278……………...………57
Figure 4.3 - β-glucuronidase activities for riboswitches with and without the extended 5'-
UTR in E. coli…………………………………………………………………………....59
Figure 4.4 - 3' end of S. pyogenes 16S rRNA aligned with the RBS of riboswitches D and
E……………………………………………………………………………..…………...60
Figure 4.5 - Theophylline-dose-dependent increase of β-glucuronidase activity……….62
Figure 4.6 - The extended 5'-UTR found downstream of the Psag promoter allows for
more robust expression of β-glucuronidase……………………………………………...63
Figure 4.7 - β-glucuronidase activity of cultures harvested at mid exponential and
stationary phases of growth……………………………………………………………...64
Figure 4.8 - The effects of cell lysate storage conditions on β-glucuronidase activity…65
Figure 4.9 - β-glucuronidase activities for riboswitches D and E in S. pyogenes……….66
Figure 4.10 - Theophylline-dose-dependent increase of β-glucuronidase activity using
improved protocol…………………………………………………………………….…67
Figure 4.11 - β-glucuronidase activities for several riboswitches in S. pyogenes………68
Figure 4.12 - Alignment of S. pyogenes 16S rRNA to riboswitch sequences…………..68
Figure 5.1 - Theophylline-dependent synthetic riboswitches controlling lacZ in
F. novicida………………………………………………………………………….……81
Figure 5.2 - Alignment of F. novicida 16S rRNA to riboswitch sequences……….……82
Figure 5.3 - Dose-dependent riboswitch-mediated induction of β-galactosidase……....83
Figure 5.4 - Riboswitch-mediated control of GFP in F. novicida during macrophage
infection………………………………………………………………………………….84
Figure 5.5 - Riboswitch E controlling FTN_0818 in minimal media…………………...86
Figure 5.6 - Riboswitch F controlling FTN_0818 in minimal media…………………....87
Figure 5.7 - Riboswitch-mediated control of FTN_0818 facilitates intracellular
replication………………………………………………………………………………..88
Figure 5.8 - Riboswitch E controlling β-galactosidase activity in F. novicida and
F. tularensis…………………………………………………………………………..…91
Figure 5.9 - Correlation of theophylline dose to theophylline concentration in blood
serum…………………………………………………………………………………….94
List of Tables
Table 5.1 - Bacterial strains and plasmids used in Chapter 5…………………………...97

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