Single-cell-level Studies of Phenotypic Diversity in Bacteria Open Access

Simsek, Emrah (Spring 2019)

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Bacteria have an amazing ability to adapt to unfavorable conditions, which is why they are all over the Earth. Population diversification is a well-known mechanism for adaptation. Importantly, an isogenic population can exhibit phenotypic diversity without changing their genotypes. My dissertation aims at elucidating the origins and implications of phenotypic diversity under three prevalent environmental conditions; nutrient fluctuations, antibiotic exposure, and restricted motility due to surface association.

We first characterized at the single-cell resolution the metabolic activities and growth kinetics of starved Escherichia coli cells subject to nutrient upshift. We observed a subpopulation of cells which resumed the growth long after the upshift. By characterizing their metabolic states, we showed that they exhibit active substrate uptake and catabolism but inactive anabolism. We showed that oxidative stress is an innate factor leading to these partial metabolic activities. We observed that cells with partial metabolic activities spontaneously restored their anabolism and grew. This growth resumption indicates that these cells were not dead but dormant.

We next quantified the temporal dynamics of the growth recovery of dormant cells in the face of bactericidal antibiotics. When a genetically-identical population faces a bactericidal antibiotic, a majority of cells dies quickly but a small fraction called persisters survives. Dormancy is the generally accepted mechanism for emergence of these persisters. When we characterized the time points at which these persister cells exit from dormancy, we found their temporal probability distribution exhibits a power-law decay. We explained this power-law decay by using heterogeneous Poisson processes. We showed how this explanation is consistent with a myriad of biomolecular pathways previously identified for entering and exiting dormancy.

Finally, we developed two visualization tools for studying the origins and implications of non-genetic population diversification in the context of surface associated-life of bacteria. These tools enabled characterization of surface-associated Proteus mirabilis bacteria at a wide range of scales from the single cell (mm) to the colony level (cm). Applying these tools to a few examples, we demonstrated how these tools could be useful.

Table of Contents

Chapter 1: Introduction. 1

1.1. Motivation. 1

1.2. Methodology. 4

1.2.1. The plate count method. 5

1.2.2. Optical microscopy and fluorescent reporters for studying living single cells. 7

1.2.3. Mathematically representing cellular processes using ordinary differential equations. 10

1.3. Outline of Dissertation. 14

Chapter 2: The emergence of metabolic heterogeneity and diverse growth responses in isogenic bacterial cells 17

2.1. Abstract. 17

2.2. Introduction. 18

2.3. Results. 20

2.3.1. Cell-to-cell heterogeneity in metabolic activities and growth phenotypes. 20

2.3.2. Emergence of dormant cells with partial metabolic activities. 34

2.3.3. Oxidative stress induces the emergence of dormant cells with partial metabolic activities. 38

2.4. Discussion. 44

2.5. Methods. 47

2.6. Movie Caption. 55

Chapter 3: Power-law tail in lag time distribution of bacteria: its origin and implication for bacterial persistence in the face of antibiotics 57

3.1. Abstract. 57

3.2. Introduction. 58

3.3. Results and Discussion. 60

3.3.1. Multiphase population dynamics of isogenic bacteria exposed to an antibiotic. 60

3.3.2. Single-cell-level observation of lag time. 62

3.3.3. Time delay of ampicillin killing. 65

3.3.4. Mathematical framework bridging lag time distribution and time-dependent killing curve. 67

3.3.5. Quantitative mechanisms for exponential or power-law decays in rejuvenation probability. 70

3.3.6. Power-law decay provides a quantitative framework for understanding complex molecular processes underlying persistence. 72

3.3.7. Further implication of a power-law decay. 73

3.4. Methods. 74

3.5. Appendix. 79

Chapter 4: Developing visualization tools for studying surface colonization by Proteus mirabilis across multiple scales 82

4.1. Abstract. 82

4.2. Introduction. 83

4.3. Representative Results. 85

4.3.1. Spatiotemporal characterization of flhDC promoter activity at the single cell level using a transcriptional fluorescent reporter. 85

4.3.2. A multiscale analysis of the onset of swarming. 89

4.3.3. In situ visualization of the motion of individual swarmer cells. 92

4.4. Discussion and Future Directions. 94

4.5. Materials and Methods. 99

4.5.1. Construction of a P. mirabilis strain with a single chromosomal copy flhDC-gfp transcriptional fusion. 99

4.5.2. A markerless in-frame deletion of the rcsC gene in the P. mirabilis strain with a single chromosomal copy flhDC-gfp transcriptional fusion. 100

4.5.3. Construction of a plasmid allowing a constitutive high expression of a green fluorescent protein gene. 101

4.5.4. Bacterial growth conditions. 102

4.5.5. Strain and plasmid construction. 104

4.5.6. Microscope imaging and analysis. 105

4.5.7. Development of a Petri dish based-device for multiscale studies of swarming. 106

Chapter 5: Summary and Outlook. 108

References. 117

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