Pilizota, Teuta

Nakayama, Naomi

Hegde, Smitha Seetharama

2020-03-09T13:15:59Z

2020-03-09T13:15:59Z

2020-07-04

Water is central for all living cells; in prokaryotes—also in fungi and plants— osmotic forces regulate the water available for cellular functions. The osmotic pressure inside a cell arises due to the higher concentration of cell membrane impermeable solutes inside the cell than its external environment. During any sudden perturbations in the environmental osmolarity, the primary implications on the cell is swelling or dehydration. The subsequent response of the cell, in the event of dehydration due to a hyperosmotic shock, is to restore the cellular water by actively increasing the solute concentration inside the cell using osmoregulatory network. In the event of swelling due to a hyposmotic shock, resulting increase in cell membrane tension triggers opening of a series of mechanosensitive channels (MSCs), which opens pores through which the solutes and cellular water diffuse out. These two diverse osmoregulatory mechanisms adjust the internal osmotic pressure in a bacterium to maintain a pressure homeostasis. To study osmoregulation, bacteria which live in the gut of animals make an ideal system, as they experience a constantly changing external environment due to the complex feeding habits and metabolic activity of their hosts. And among these bacteria, Escherichia coli is one of the simplest and best understood organisms. With a curiosity to understand life in the context of survival to osmotic challenges, in this thesis, I explore the single cell responses of E.coli to hyposmotic shocks. Using epi-fluorescence microscopy and constitutively expressing eGFP as cytoplasm marker, I first characterize the in-vivo volume responses of the wildtype E.coli to a hyposmotic shock. The characteristic volume response includes fast volume expansion due to water influx and a subsequent slower volume recovery through MSCs, which also overshoots below the initial volume. The fast volume expansion is on the order of 0.5-2s whereas the recovery phase lasts few minutes. To affirm that the volume recovery is through MSCs, I next measure the volume response in a double and hepta MSCs deletion mutants. These two mutants exhibit a fast volume expansion but not the characteristic volume recovery of the wild-type. The double mutant shows a small volume recovery and hepta-mutant remains swollen throughout the duration of imaging (up to few hours). For a large 960 mOsmol hyposmotic shock, ≈ 36 % of the hepta mutant cells survive the challenge. A closer look into the death of hepta-mutant suggests that the quality of cell attachment to the coverslip influences the time of death, poorly attached cells dying sooner. In this thesis I study the dynamics of cell death due to hyposmotic shock and discuss the possible influence of cell-wall mechanics on the survival of the hepta mutant. In contrast to membrane embedded ion channels MSCs are non-specific to the solutes that pass through them. While this non-specificity helps bacteria restore osmotic pressure during a hyposmotic shock, it can also cause excess loss of solutes, which can also lead to volume overshoot. Given that cell’s response to hyposmotic shock is passive, I also investigated the possible additional levels of control that could fine tune E.coli’s passive response to hyposmotic shock. Specifically, I looked at co-operative gating of MSCs through clustering in the cell membrane. With a continuum phenomenological model, supported by collaborative results from a coarse-grained model of MSCs aggregation, I demonstrate that the MSCs clustering regulates the whole cell volume during a hyposmotic shock and does it in a way to reduce excessive solute loss without impeding the functionality of MSCs. In the final part of the thesis, I apply the knowledge gained on hyposmotic response of E.coli for industrial biotechnology applications. Specifically, I optimize the osmotic extraction of an industrially important periplasmic protein, Hel4, by deleting a replaceable centrifugation step without compromising on yield. Additionally, I address the leakage of the periplasmic Hel4 in industrial fermentation by looking at the role of outer-membrane porins, OmpC and OmpF.

https://hdl.handle.net/1842/36844

http://dx.doi.org/10.7488/era/146

en

The University of Edinburgh

Renata Buda, Yunxiao Liu, Jin Yang, Smitha Hegde, Keiran Stevenson, Fan Bai, and Teuta Pilizota. Dynamics of escherichia coliâAZs passive response to a sudden decrease in external osmolarity. Proceedings of the National Academy of Sciences, 113(40):E5838–E5846, 2016.

fluorescence microscopy

Escherichia coli

E. coli

osmotic pressure

single cell imaging

mechanosensing

bacteria

periplasmic protein extraction

Escherichia coli’s response to hyposmotic shocks

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy