Investigating the Key cell division proteins FtsZ and SepF in Streptomyces coelicolor

Clark, Lucy (2022) Investigating the Key cell division proteins FtsZ and SepF in Streptomyces coelicolor. Doctoral thesis, University of East Anglia.

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Understanding the regulation of cell division in Streptomyces coelicolor is key to gaining insight into how the complex life cycle of this bacterium is regulated. To date, researchers have established a key cell division protein FtsZ, yet little is known as to how its function is controlled and which proteins associate with FtsZ to form septum. In this thesis I therefore investigate several proteins in S.coelicolor to gain a better understanding of their role in cell division. In Chapter 3 I used the basic binding assay; Bacterial Two Hybrid, to test Interactions between Proteins involved in Cell division, Polar growth and DNA Segregation. Whilst known protein interactions were confirmed; many new binding partners were discovered between a range of proteins involved in all stages of the life cycle. This included confirming SepF2 can bind the divisome initiating protein FtsZ. Futhermore, SepF2 was shown to bind cytokeneisis protein SsgB and polar growth protein ParH. In Chapter 4 I present the initial characterisation of the potential SepF homologs to investigate their role within S.coelicolor cell division. I use bioinformatics and in vitro techniques to characterise SepF2. SepF2 shares great sequence identity to its homologs but is not predicted to be able to bind lipid membranes. When viewed using microscopy, It forms polymers of long coil structures which take on the appearance of a spring. In Chapter 5 I attempt to gain a better insight into the biochemical properties of FtsZ from S.coelicolor. I use bioinformatics to generate a three dimensional model of FtsZ which shares conserved features with homologs in other bacterium. I use protein assays to understand the dynamics and polymerisation properties of FtsZ. The protein is able to self associate using the ligand Magnesium and requires Guanine Tri Phosphate (GTP) for disassembly of polymers. In Chapter 6 I characterise SepF2 (SCO2079) a potential SepF homolog found within the cell division gene cluster. I generate a three dimensional structure of the C-terminal domain of SepF2 which appears to share key residues required for FtsZ binding and dimer formation with its homolog in Bacillus subtilis. Using Analytical Gel Filtration and buffer optimisation I stabilise the formation of a truncated variant of SepF2 into dimers. Overall, I have found that SepF2 in S.coelicolor appears to play a crucial role in several stages of the bacterium’s life cycle as it binds to proteins linked to growth, cell division and DNA segregation. The work reported in this thesis contributes to improving our understanding around the regulation of the life cycle of S.coelicolor, in particular gaining insight into two key proteins FtsZ and SepF2.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Kitty Laine
Date Deposited: 05 Dec 2022 10:44
Last Modified: 05 Dec 2022 10:44

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