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Uncovering the formation of the Gram-negative bacterial cell envelope, and peptidoglycan synthesis
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Graham, Christopher L. B. (2023) Uncovering the formation of the Gram-negative bacterial cell envelope, and peptidoglycan synthesis. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3977730
Abstract
This thesis is about how bacteria grow, especially how they grow the cell wall that keeps them rigid, and their cell envelope. Specifically, this thesis is on understanding the formation of the Gram negative version of the bacterial cell envelope, which constitutes a great deal of pathogenic species. Gram-negative bacteria are a sub-class of bacteria within the phylogeny of prokaryotes (1). Bacteria like all living organisms are formed partly of a DNA core or chromatin, with central processes that allow for propagation of this chromatin, encapsulated by a cellular envelope. The cell envelope in Gram-negative is a layer of protection including a cell wall with fatty membranes on either side, including especially an outer membrane(2). My PhD project on these bacteria has explored the coordination and creation of this cell envelope, including the creation and destruction of bacteria peptidoglycan or glycan sugar mesh which gives this envelope structure (Chapters 1- 3). In addition to this it looks at the lipid/fatty part of the cell envelope, and postulates parts of its formation (Chapter 4). Phospholipid layer biosynthesis systems create the fatty membranes that give the envelope flexibility and its ability to separate the ions and other small molecules within the organism from the environment, this is essential for life. The aim of this thesis is to improve understanding of Gram-negative (aka diderm) bacterial cell growth and division with a notion to use this understanding for future antibiotic treatment. Our central hypothesis is that peptidoglycan (PG) and outer membrane (OM) cell envelope metabolism systems must be linked. This combined study is essential, as Gram-negative bacteria make up some of our most relevant pathogens, with more deaths caused by these organisms and their resistance to treatment, than AIDs and Malaria (3). Understanding the cell envelope will allow us to combat these organisms.
The starting point of this thesis came from our studies working on expanding the knowledge of the current core of the ‘elongasome’ responsible for cell growth; RodA and PBP2 (4,5), which have been shown to together increase the chain length of glycan strands, required for longitudinal cell wall extension (6). I started with an aim to link this core complex with hypothesized partners LpoA, PBP1a and other proteins with potential regulatory or enzymatic roles. However upon bioinformatic analysis (2)(Chapter 1) I realised this is more complex and likely involves systems which create the entire cell envelope, and thus the scope of the PhD initially imagined as Chapter 2’s topic on RodA-PBP2 was widened beyond the initial RodA-PBP2 complex. The Chapter 1 background along with methods established in Chapter 2, (which use in vitro assays of the RodAPBP2 system to create polymerised lipid II, and find the mechanism of this enzymes action ) are then used to create a new assay to uncover function of the cell wall breakdown enzymes the lytic transglycosylases in Chapter 3. In Chapter 4 I took the understanding gained from the interaction of the SEDS machinery with other proteins, and the connection to outer membrane proteins uncovered in Chapter 1, 2 and 3 to postulate and see under the microscope the localisation of outer membrane phospholipid insertion (7,8) and biosynthetic machines (MlaD/PqiB/YebT/etc) in the cell envelope (10), and use a phosphatidylcholine mimic only usable in Pseudomonas aeruginosa in Chapter 4 to study lipid movement and localisation. This was in the hopes of finding new patterns in outer membrane and cell envelope laydown, which may follow similar trends to that of peptidoglycan laydown established in Chapter 1 and 2. I summarise this work, and the linked papers in each chapter’s conclusion and my thesis’s impact in the ‘Thesis conclusion’.(9) Finally, whilst doing my PhD which explores the network of proteins in Gram-negative cell envelopes, I also wrote three papers on creating communities in science, which acknowledge the thesis, and hypothesise new tools for combatting the future of AMR internationally in a changing world.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QH Natural history Q Science > QR Microbiology Q Science > QR Microbiology > QR180 Immunology |
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Library of Congress Subject Headings (LCSH): | Nuclear membranes, Gram-negative bacteria, Peptidoglycans -- Synthesis, Bacterial cell walls, Membrane lipids, Cells -- Growth, Drug resistance in microorganisms | ||||
Official Date: | March 2023 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Roper, David I. | ||||
Sponsors: | University of Warwick. School of Life Sciences ; Biotechnology and Biological Sciences Research Council (Great Britain) ; Antibiotic Research UK ; Natural Environment Research Council (Great Britain) ; MITACS (Network) | ||||
Format of File: | |||||
Extent: | multiple pagings : coloured illustrations | ||||
Language: | eng |
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