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Understanding and imaging early stages of bacterial cell division in Gram-positive pathogens

Principal Supervisor: Professor David I Roper, School of Life Sciences

Co-supervisor: Professor Tim Dafforn, Scholl of Biosciences, U. Birmingham

PhD project title: Understanding and imaging early stages of bacterial cell division in Gram-positive pathogens

University of Registration: University of Warwick

Project outline:

Bacterial cell division is orchestrated by a group of membrane bound and cytoplasmic proteins, which form a complex known as the Divisome. In order for the cell to divide correctly, the division process must be coordinated with biosynthesis of new cell wall material and separation of daughter chromosomes mediated by specific divisomal protein sub-complexes. Arrest of these events leads to the cessation of normal growth or cell lysis and is of interest therefore from both a basic scientific and translational perspective. All bacterial undergo this process and it is a conserved and evolutionary related process to many other forms of life and cellular activity.

Most work in the literature concerns Gram-negative E. coliand Gram-positive B. subtiliswhich are both rod shaped bacterial which have both divisomal and elongase complexes required for growth and maintenance of rod shape. In contrast to other rod-shaped bacteria, S. aureusforms spherical cells with a minimal genetic and protein complement required for cell division and thus in many ways provides an elegant minimal system in which to study and understand cell division from first principles.

This project will focus on understanding the interplay between the bacterial divisomal components in early stage Gram-positive bacterial division. We will focus on S. aureusas a model system and in particular the central complex formed between the cytoplasmic anchor protein: EsrA, and the divisomal proteins FtsZ and FtsA. These proteins provide a cytoplasmic and bacterial inner membrane linkage to the enzymes outside the cell, responsible for biosynthesis of the bacterial cell wall peptidoglycan. These proteins, known as penicillin binding proteins (PBPs) are well characterised antimicrobial drug targets, which in S. aureus are PBP1 (class B: monofunctional transpeptidase and PBP2 (Class A: bifunctional glycosyltransferase and transpeptidase) are the key proteins. Coordination of these activities is a key process in cell division and cell wall biosynthesis. How these proteins are physically linked and coordinated through the membrane is largely unknown. Previous collaborative work from our laboratories using a styrene maleic acid polymer (SMALP) nanodisc reagent enabled the isolation of S. aureus PBP2 and PBP2a (MecA) from MRSA cells and we will now extend this analysis to investigate the nature and coordination of these interactions. Importantly this technique allows the isolation of the proteins in the presence of membrane native lipids in contrast to conventional detergent based isolation techniques for membrane proteins which remove the native lipidic environment and we have pioneer the development of this technique and its application to bacterial cell division.

This project will involve close collaboration between the Roper group at Warwick and the Dafforn group at Birmingham who have been working together on cell division for some time. The student will have access to facilities and expertise in both laboratories to enhance and maximise the scientific potential and development of the project.

In this project we will address the following biological questions.

  1. What are the essential elements of the S.aureus divisomal protein complex and how are they arranged with respect to each other?
  2. How do these proteins exert a contractile force on the membrane that leads to cell division?
  3. What influence do membrane lipids have on the complex and does the membrane alter in lipid composition during constriction?

BBSRC Strategic Research Priority: Molecules, Cells and Systems

Techniques that will be undertaken during the project:

Microbiology, molecular biology including the creation of genetically modified bacterial strains biochemistry including membrane protein isolation and purification, small scale chemical preparation of styrene malic acid polymer (acid reflux and freezer drying), biophysical analysis using analytical ultracentrifugation, small angle X-ray scattering, negative stain and cryo electron microscopy and live cell imaging of fluorescent labelled proteins in S. aureus.

Contact: Professor David Roper, University of Warwick