Date of Graduation

5-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Microelectronics-Photonics (PhD)

Degree Level

Graduate

Department

Microelectronics-Photonics

Advisor/Mentor

Wang, Yong

Committee Member

Muldoon, Timothy J.

Second Committee Member

Li, Jiali

Third Committee Member

Chen, Jingyi

Fourth Committee Member

Leftwich, Matthew B.

Keywords

Bacterial cytoplasm; single particle tracking photoactivation localization microscopy (sptPALM); E. coli bateria; H-NS proteins

Abstract

Bacteria are small organisms that play important roles in our bodies and in maintaining our surrounding environment. However, bacterial infections are considered as one of the health diseases that threatens the lives of humans. Resistance of bacteria to antibiotics has made treating bacterial infections more challenging. With the emergence of many developed optical techniques, it has become possible to study various biological mechanisms of molecules inside these small organisms with high resolution and precise localization. In this research, single particle tracking photoactivation localization microscopy (sptPALM) technique was used to investigate the dynamics of fluorescent histonelike nucleoid-structuring proteins (H-NS) in live E. coli bacteria at normal conditions and different environmental conditions. It was found that the distribution of the diffusion coefficients of H-NS proteins was a power law. Moreover, the distribution of displacements of H-NS proteins followed a Pearson Type VII distribution rather than other distributions. Furthermore, the viscoelasticity of the bacterial cytoplasm was measured experimentally by calculating the complex modulus of the cytoplasm as a function of frequency. Accordingly, a transition was observed in the viscoelasticity of bacterial cytoplasm from a glass-like structure into liquid-like structure in the frequency domain. Moreover, the diffusive dynamics of H-NS proteins were probed in different lengths of bacterial cell. It was found that the diffusions of H-NS proteins became faster as the lengths of the bacterial cells increased. In this work, the effect of Ag+ ions on the diffusive dynamics of H-NS proteins in live bacterial cells were studied. The diffusions of H-NS proteins in live bacteria increased as the treatment time with Ag+ ions increased. In vitro technique, electrophoretic mobility shift assay (EMSA), was done to understand the reasons behind the faster motions of H-NS proteins upon the treatment with Ag+ ions. The result of this technique showed that Ag+ ions affected the binding of H-NS proteins to the DNA and made it weaker. Furthermore, the isothermal titration calorimetry technique was used to probe the direct interaction between the DNA and Ag+ ions. Bent DNA molecules were used to study the effect of Ag+ ions on the double-stranded DNA. The results showed that Ag+ ions interacted with DNA and caused a dehybridization of the double-stranded DNA into single-stranded DNA. Lastly, the effect of temperature stress on the diffusive dynamics of free cytoplasmic particles and binding proteins (H-NS) proteins in live and dead cells using (sptPALM) technique was studied. It was found that the diffusive dynamics of H-NS proteins became faster as the temperatures increased from 21 to 37 °C. Importantly, it was found that the non-thermal energy (ATP) contributed to the changes in the diffusion of H-NS proteins rather than the thermal energy in dead and live bacterial cells. Different results with the effect of temperature stress on the diffusive dynamics of free cytoplasmic particles were observed. It was found that the diffusion of the cytoplasmic particles became slower as the temperature increased from 21 to 37 °C, suggesting the effect of temperature on the viscosity of the bacterial cytoplasm.

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