Single-molecule fluorescence imaging of bacterial chromosome segregation dynamics in cell-free systems
Lead Research Organisation:
University of Sheffield
Department Name: Molecular Biology and Biotechnology
Abstract
Cell division is a process of fundamental importance to all forms of life. During cell division, DNA must be properly segregated into each daughter cell in order to ensure the stable inheritance of genetic material. The aim of this project is to gain insight into the molecular mechanisms underpinning bacterial chromosome segregation from Vibrio cholerae as a target for novel antibiotics.
The process of chromosome segregation in bacteria is regulated by Par (partition) proteins. Recent advances in fluorescence microscopy have revealed that bacterial chromosomes are highly organized and segregate with distinctive patterns in the cell. In this project, model systems consisting of nanopatterned arrays of biomolecules will be constructed and used as biomimetic platforms. These platforms will be utilized for the study of dynamics of partition proteins from Vibrio cholerae in order to better understand how they are involved in chromosome segregation. This project combines multidisciplinary approaches including single-molecule fluorescence microscopy, biochemistry, and bionanotechnology to visualize the purified proteins on synthetic surfaces in a cell-free system.
The process of chromosome segregation in bacteria is regulated by Par (partition) proteins. Recent advances in fluorescence microscopy have revealed that bacterial chromosomes are highly organized and segregate with distinctive patterns in the cell. In this project, model systems consisting of nanopatterned arrays of biomolecules will be constructed and used as biomimetic platforms. These platforms will be utilized for the study of dynamics of partition proteins from Vibrio cholerae in order to better understand how they are involved in chromosome segregation. This project combines multidisciplinary approaches including single-molecule fluorescence microscopy, biochemistry, and bionanotechnology to visualize the purified proteins on synthetic surfaces in a cell-free system.
Organisations
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| EP/N509735/1 | 01/10/2016 | 30/09/2021 | |||
| 1903415 | Studentship | EP/N509735/1 | 01/11/2016 | 21/06/2020 | Satpal Chodha |
| Description | DNA segregation is essential in all cell types. In eukaryotes, spindle fibres pull apart replicated chromosomes to ensure a copy is faithfully transmitted to each daughter cell. While fluorescence microscopy has given insight into the choreography of chromosomes during a cell cycle, the exact mechanism is still unknown. It has been found however that ~70% of bacterial chromosomes encode a partitioning system called a parABS system. This system is also present on bacterial plasmids (DNA separate from genome), where much research has been done. The diffusion-ratchet model is the most likely model to account for plasmid partition. We now want to investigate if a similar model exists for chromosomal parABS systems. The usual workhorse organism, E. coli, does not encode a parABS system, and it's not known how it preforms chromosome segregation. Vibrio cholerae is closely related to E.coli, and has 2 chromosomes, each with a distinct parABS system for good measure. It turns out the secondary chromosome parABS system is closely related to plasmid systems so this is the model system in this research. I am currently in the process of comprehensively biochemically characterising the secondary chromosome segregation protein from Vibrio cholerae, ParA2. ParA2 is a DNA binding ATPase, which acts as the motor protein to drive bacterial chromosome segregation. This will be one of the first chromosomal ParAs to be characterised to this extent. DNA binding, ATP binding kinetics, ATP hydrolysis, and the effects of ATP binding have all been investigated. There are similarities and distinctions between plasmid systems, and the significance of these results will be investigated. Fluorescence microscopy of cell-free reconstitutions will next be utilised to look into ParA2 interacting with other components of the parABS2 system: ParB2, and parS2-DNA. This will determine the mechanism of action of the parABS system of VC chrII. |
| Exploitation Route | There are no ParA homologues in eukaryotes, making it an ideal target for antibiotics. |
| Sectors | Pharmaceuticals and Medical Biotechnology |