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dc.contributor.advisorPilizota, Teutaen
dc.contributor.advisorEl Karoui, Meriemen
dc.contributor.authorMiroli, Darioen
dc.date.accessioned2020-04-01T12:47:28Z
dc.date.available2020-04-01T12:47:28Z
dc.date.issued2020-07-04
dc.identifier.urihttps://hdl.handle.net/1842/36931
dc.identifier.urihttp://dx.doi.org/10.7488/era/232
dc.description.abstractThe goal of the study of bacterial physiology, and in the study of biophysics more broadly, is to apply quantitative predictive models to the behaviour of complex living organisms. Just as is the case with thermodynamics, the challenge comes from the fact that the subject systems are far too intricate to be modelled from the bottom up using first principles. Instead, the focus is on the development of coarse-grained models which reduce the description of the system down to a small number of key state variables. As late as the mid 20th century the identity, and even existence, of these variables was an open question, because living cells are a non-equilibrium system. However, between the 1950s and 1970s a golden era of bacterial physiology would give rise to a series of robust quantitative models, that linked a number of important physiological parameters. Starting with the Copenhagen school in 1958, it would soon be discovered that cell size was connected to growth rate via a single exponential relation that was independent of the makeup of the media used to achieve it. Shortly after this key result, the work of Helmstetter and Cooper would reveal that the time taken to replicate the chromosome of E. coli was constant over a wide range of growth rates. This counter intuitive observation would lead to the realisation that in order to grow at doubling times shorter than their replication time, bacterial cells must overlap multiple rounds of replication simultaneously. Taking these findings and running with them, Donachie at the University of Edinburgh was able to develop a model in which cells accumulate a critical initiation mass in order to start new rounds of replication. Using previous data, he was able to show that this initiation mass remained constant across a wide range of growth conditions, and in doing so, was able to relate neatly the average cell size, growth rate, chromosome replication time and time to divide. Since its development Donachie’s constant initiation mass model has been tested under numerous different environmental conditions and found to hold. Although examples exist where the initiation mass can be shown to vary, the underlying relationship continues to stand up to scrutiny. Despite this extensive testing, one condition underrepresented in literature is that of growth at high osmolarity. Much like nutrient limitation, high osmolarity conditions have been shown to reduce the growth rate although the mechanism by which this happens remains poorly understood. To this end the objective of this doctoral thesis is to examine the balanced growth of E. coli cells in high osmolarity conditions, and determine how the cell physiology under such conditions fits within the paradigm of constant initiation mass, as described by Donachie. To achieve this, I characterise the bulk steady state growth rates of cells and show that in agreement with previous work they decrease significantly with increasing salt concentration. I go on to measure cell volume both using a custom built microscope I constructed, and a high throughput Coulter counter. By comparing the results from these independent measurement I am able to show that, in steady state, there is very little volume change that accompanies the significant decrease in growth rate at high osmolarity. Following the analysis of cell size, I proceed to measure how the DNA content of cells changes at high osmolarity, using flow cytometry. I argue that, in contrast to some recent findings, there is little change in cellular DNA content as the salt concentration is increased. Combining these results with measurements of the chromosome replication time from real time PCR reactions, I also demonstrate an increase in the cell cycle time with increasing osmolarity, in agreement with recent findings. Combining these results, I am able to show what effect high osmolarity has on the initiation mass. Additionally, I examine the results from a small number of experiments where the osmolarity was increased using soribtol instead of sodium chloride. I discuss if the nature of the osmolyte used changes the effect a high osmolarity condition has on cellular physiology, and suggest some interesting experiments going forward to further understand this relationship.en
dc.language.isoen
dc.publisherThe University of Edinburghen
dc.subjectE. colien
dc.subjectosmolarityen
dc.subjecthigh osmolarityen
dc.subjectconstant initiation massen
dc.subjectcell volumeen
dc.titleCell size regulation of Escherichia coli at high osmolarityen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen
dc.rights.embargodate2021-07-04
dcterms.accessRightsRestricted Accessen


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