Transcriptional and post-transcriptional mechanisms of thermoregulation in Pseudomonas aeruginosa Restricted; Files Only
Robinson, Rachel (Fall 2025)
Abstract
Pseudomonas aeruginosa is an opportunistic pathogen of great medical and public health importance. P. aeruginosa can cause chronic and even life-long respiratory infections in people with the genetic disorder cystic fibrosis. It is frequently isolated from human-associated environments such as hospital surfaces and is a common nosocomial pathogen that is notoriously difficult to treat due to its high intrinsic and often acquired antibiotic resistance; extensively drug-resistant strains have also emerged that are resistant to all clinically available antibiotics. Nosocomial transmission of this bacterium involves transitioning from a room temperature environment (~25°C) to one more associated with the human body (~37°C). Understanding thermoregulation of cellular processes is thus important for uncovering how P. aeruginosa adapts to the human body, particularly during the initial stages of infection, yet this topic remains understudied. We used RNA-sequencing to determine the global thermoregulon of P. aeruginosa at exponential and stationary phases and characterized growth-phase dependent differences in thermoregulation. We found many quorum sensing regulated genes were thermoregulated and determined the regulon of LasR, the master quorum sensing regulator, at 37°C and 25°C. The transcriptomes generated as part of this dissertation provide an important resource for the P. aeruginosa field. We also investigated the mechanistic basis of thermoregulation of two genes arising from RNA-sequencing experiments: piv and capB. We show that the gene encoding protease IV (piv) is thermoregulated only at stationary phase and its expression is significantly higher at 25°C than 37°C. This is due to temperature-dependent promoter activity resulting from increased upregulation of piv by LasR at 25°C than 37°C. As PIV is a known virulence factor, we also investigated the role of piv in temperature-dependent virulence of P. aeruginosa using the model organism Galleria mellonella. capB is a putative cold shock RNA chaperone that is highly upregulated at 25°C. We show that the capB transcript has a long 5’ untranslated region (UTR) and that this UTR is important for post-transcriptional thermoregulation of capB that results in higher protein levels at low temperatures. These studies have enhanced our mechanistic understanding of how the important pathogen P. aeruginosa senses and adapts to changes in temperature.
Table of Contents
TABLE OF CONTENTS
Chapter One: Introduction: Mechanisms of thermoregulation in Pseudomonas aeruginosa 1
The importance of Pseudomonas aeruginosa, an opportunistic human pathogen.............. 2
How do bacteria sense, and respond to, temperature changes?......................................... 4
‘Omics Studies of Thermoregulation..................................................................................... 7
Transcriptional Thermoregulation.......................................................................................... 9
Post-Transcriptional Thermoregulation............................................................................... 14
Post-Translational Thermoregulation.................................................................................. 19
Outstanding questions of P. aeruginosa thermoregulation................................................. 21
References........................................................................................................................... 23
Chapter Two: The interplay between temperature and growth phase shapes the transcriptional landscape of Pseudomonas aeruginosa................................................................................................................................................. 40
Abstract................................................................................................................................ 41
Introduction.......................................................................................................................... 42
Results................................................................................................................................. 45
Discussion............................................................................................................................ 65
Materials and Methods........................................................................................................ 71
Acknowledgements.............................................................................................................. 73
References........................................................................................................................... 74
Chapter Three: Temperature controls LasR regulation of piv expression in Pseudomonas aeruginosa 124
Abstract.............................................................................................................................. 125
Introduction........................................................................................................................ 126
Results............................................................................................................................... 129
Discussion.......................................................................................................................... 143
Materials and Methods...................................................................................................... 149
Acknowledgments.............................................................................................................. 154
References......................................................................................................................... 155
Supplemental Material....................................................................................................... 161
Chapter Four: piv does not impact Pseudomonas aeruginosa virulence in Galleria mellonella 176
Abstract.............................................................................................................................. 177
Introduction........................................................................................................................ 178
Results and Discussion..................................................................................................... 181
Materials and Methods...................................................................................................... 190
Acknowledgments.............................................................................................................. 194
References......................................................................................................................... 195
Supplemental Material....................................................................................................... 200
Chaper Five: Post-transcriptional thermoregulation of capB by its own 5’ untranslated region 202
Introduction........................................................................................................................ 203
Results............................................................................................................................... 206
Discussion.......................................................................................................................... 216
Materials and Methods...................................................................................................... 219
References......................................................................................................................... 229
Chapter Six: Discussion on and future directions of thermoregulation in Pseudomonas aeruginosa 235
Global thermoregulation in Pseudomonas aeruginosa..................................................... 236
The intersection of LasRI quorum sensing and thermoregulation in P. aeruginosa......... 239
The cold shock RNA chaperone CapB.............................................................................. 241
Concluding remarks........................................................................................................... 243
References......................................................................................................................... 245
TABLE OF FIGURES
Chapter One
Figure 1. P. aeruginosa traverses environments of different temperatures as an opportunistic and nosocomial pathogen. 4
Chapter Two
Figure 1. Temperature regulates the expression of hundreds of distinct genes in P. aeruginosa at both exponential and stationary phases. 53
Figure 2. Metabolic pathways upregulated in P. aeruginosa growing at 37°C at exponential phase. 55
Figure 3. capB is the only putative cold shock response gene regulated by temperature..... 56
Figure 4. Growth phase regulates genes similarly at 25°C and 37°C.................................... 58
Figure 5. LasR regulates most target genes similarly at 25°C as 37°C, with notable exceptions. 63
Chapter Three
Figure 1. Temperature regulation of piv depends on growth phase..................................... 130
Figure 2. Temperature regulation of piv expression is reflected by levels of PIV protein..... 132
Figure 3. Temperature alters activity of the piv promoter through transcriptional regulator LasR, but not MvaT/MvaU. 136
Figure 4. LasRI quorum sensing regulation is not higher at 25°C than 37°C....................... 138
Figure 5. Mutations to the piv promoter region alter promoter activity.................................. 141
Figure 6. A proposed model for the transcriptional thermoregulation of piv by the quorum sensing regulator LasR. 145
Supplemental Figure 1. Background fluorescence from a no-promoter gfp(ASV) reporter plasmid is low. 171
Supplemental Figure 2. MvaT and MvaU are not thermoregulated...................................... 172
Supplemental Figure 3. Verification of aLasR antibodies to detect LasR............................ 173
Chapter Four
Figure 1. Mature PIV VSV-G protein levels in P. aeruginosa supernatants are thermoregulated. 182
Figure 2. Schematic of G. mellonella larvae infection by P. aeruginosa strains at two temperatures. 183
Figure 3. Deletion of piv does not impact virulence of P. aeruginosa in G. mellonella......... 185
Supplemental Figure 1.......................................................................................................... 200
Supplemental Figure 2........................................................................................................... 201
Chapter Five
Figure 1. capB expression and CapB protein levels are inversely related to temperature.. 208
Figure 2. The capB 5’ UTR is required for its post-transcriptional thermoregulation and CapB autoregulation. 213
Figure 3. CapB does not regulate itself transcriptionally...................................................... 215
LIST OF TABLES
Chapter Two
Table 1. Low oxygen response genes that are thermoregulated............................................ 83
Table 2. Genes whose LasR regulation depends on temperature.......................................... 89
Chapter Three
Table S1. Bacterial strains used in this study........................................................................ 167
Table S2. Plasmids used in this study................................................................................... 167
Table S3. Primers used in this study..................................................................................... 169
Chapter Four
Table 1. Bacterial strains used in this study.......................................................................... 190
Table 2. Primers used in this study........................................................................................ 191
Chapter Five
Table 1. Bacterial strains used in this study.......................................................................... 219
Table 2. Plasmids used in this study...................................................................................... 219
Table 3. Primers used in this study........................................................................................ 220
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