Thesis
Imaging and quantification of nutrient-transporting channels in Escherichia coli biofilms
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2024
- Thesis identifier
- T16853
- Person Identifier (Local)
- 201994794
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- The internal structure of biofilms is crucial for their growth and development. In particular, the network of internal channels recently discovered in Escherichia coli functions as a nutrient uptake and diffusion system. While mesoscopic imaging of these channels has proven to be a powerful visualisation tool, the lack of a specialised image analysis pipeline has so far prevented an accurate quantitative characterisation of channel morphology. The genetic determinants of channel formation also remain unknown. This thesis describes the imaging and quantification of the network of nutrienttransporting channels in E. coli biofilms through open-source image analysis methods, and presents an experimental framework to perform gene deletions leading to a change in cell shape for the E. coli strain JM105 mini-Tn7-gfp. The morphology of bacterial biofilms is strongly affected by environmental growth conditions, with mechanical and biochemical composition of the growth substrate contributing to the formation of complex three-dimensional patterns. Thanks to the combination of mesoscopic fluorescence imaging and open-source image analysis, the width of individual nutrient-transporting channels was measured for biofilms formed by the E. coli strain JM105 mini-Tn7-gfp, revealing a strong dependence of channel width on both spatial location inside the biofilm and nutrient availability within the substrate. The mechanism of nutrient transport within channels was proposed to follow fluid dynamic behaviour, which would lead to increased nutrient flow towards the centre of the biofilm, where channels are smaller in diameter. The use of fractal geometry tools for the quantification of biofilm morphology and expansion patterns is well documented. The network of intra-colony channels in E. coli was also originally predicted to exhibit a fractal morphology, and this was verified in this work through fractal analysis of mature E. coli biofilm images. A dependence of channel architecture on E. coli cell shape was hypothesised due to channel formation being an emergent property of biofilm formation, and this was investigated with four cell shape mutants of E. coli obtained from the Keio collection, which is a single-gene knockout library derived from the laboratory strain BW25113. The complexity of internal channels was found to be comparable to that of computer-generated fractals for all strains grown on both rich and minimal medium substrates, though cell shape was not identified as a unique channel morphology descriptor. The characterisation of nutrient-transporting channels in E. coli has so far been performed on the laboratory strain JM105 mini-Tn7-gfp thanks to its compatibility with Mesolens imaging, which provides subcellular resolution across whole, multi-millimetre sized biofilms. While the results presented in Chapter 2 of this thesis were obtained with the same strain, the fractal analysis of biofilms formed by cell shape mutants described in Chapter 3 was performed on the strain BW25113, the parental strain for the Keio collection. This was due to the rapid biofilm disruption to planktonic state exhibited by BW25113 during immersion with liquid mounting medium, which was necessary to match the refractive index of the Mesolens used in water immersion mode. A genetic engineering protocol based on Lambda Red recombination was hence designed in order to circumvent this problem and carry out the inactivation of genes involved in the regulation of cell shape in E. coli JM105 mini-Tn7-gfp. While testing this method, the resistance of JM105 mini-Tn7-gfp to the antibiotic ampicillin was discovered and characterised, leading to a proposed modification of the genetic engineering protocol using traditional cloning methods.
- Advisor / supervisor
- McConnell, Gail
- Hoskisson, Paul A.
- Resource Type
- Note
- Previously held under moratorium from 13 March 2024 until 13 March 2026.
- DOI
- Date Created
- 2023
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