Architecture of the bacterial divisome.

All cell types need to grow and to divide in order to survive. Bacteria, one of the most primitive and ancient of all life forms, divide by a relatively simple mechanism, but nonetheless in a process that is tightly controlled and co-ordinated. The key steps include the replication of the cell's DNA, so that each copy of the cell has an identical copy of the genome, the blueprint of life. The two copies of the genome then have to be separated so that they are not entangled when the mother and daughter cells finally separate to become discrete entities, each with an identical copy of the genome. The division process normally takes place at the centre of the dividing cell and a molecular machine called the Z-ring acts as a belt, constricting tighter and tighter across the middle of the cell until it closes completely, pinching off the daughter from the mother cell. Imagine a small balloon, grasped between thumb and forefinger. As the forefinger bends like a pincer against the thumb, air is squeezed into the two halves of the balloon. When there is no gap between forefinger and thumb, each of the balloon halves, one in the palm of your hand and one above your fingers, are the same volume. As the Z-ring tightens, a protective layer called peptidoglycan, made of strands of carbohydrate cross-linked by short peptide fragments, has to be made afresh and deposited on the surface of the cell. This action must be co-ordinated with all aspects of the division process, otherwise the cell membrane is left uncovered and is prone to rupture, causing death of the cell.

This proposal concerns how the regulation of the closure of the Z-ring is co-ordinated with peptidoglycan synthesis, and the precise role played by a key protein known as EzrA. These activities are co-ordinated by a multi-protein assembly called the divisome, which links the Z-ring to peptidoglycan synthesis, but in a manner that is not yet understood. We have recently solved the structure of EzrA, which is surprisingly similar to a class of proteins called spectrins that, in animals, are used to link membrane-embedded proteins to the cytoskeleton. The cytoskeleton is a shape-defining structure found on the inside face of the cytoplasmic membrane in many cell types, including bacteria, comprising different proteins including actin and tubulin. The bacterial cytoskeleton also comprises actin and tubulin-type proteins, the latter of which is the key component of the contractile Z-ring. EzrA is therefore a structural and functional homologue of spectrin, as it acts to link the cytoskeleton to membrane-embedded proteins that synthesise peptidoglycan, and other membrane-embedded cell division regulators.

In this proposal we will ascertain the molecular mechanisms used by EzrA to interact with the cytoskeleton, other members of the divisome, including the enzymes that generate peptidoglycan, and key regulators of cell division. The research program will enable for detailed comparisons to be made between the cytoskeletons of bacteria and of animals, which is currently focussed on one or two proteins. A greater understanding of the relationships and the differences between the two cytoskeletons will also provide a greater understanding of the evolution of multicellular life forms from their simpler, single-celled predecessors.

Cell division in bacteria requires co-ordination the replication of the genome with the division of the cell into two. Amongst the key proteins co-ordinating cell division are the cytoskeletal proteins, FtsA and FtsZ. FtsA is the bacterial homologue of actin, which polymerizes at the membrane surface to aid the co-localisation of the tubulin homologue, FtsZ and to anchor it to the membrane. FtsZ polymerizes in the presence of GTP to localise at mid-cell to form the Z-ring, which provides the contractile force that is necessary to close the cell division septum; the free energy released on GTP hydrolysis by FtsZ drives Z-ring closure. FtsZ and FtsA also recruit other cell division proteins to mid-cell, including peptidoglycan synthases and other proteins of as yet uncharacterized function, to form the mature divisome, a macromolecular assembly that synthesizes the cell wall of the new poles during cell division.

EzrA is a key regulator of cell division. Its absence is lethal in Staphylococcus aureus, and causes cell division delays and cell morphology defects in Bacillus subtilis. We have recently solved the crystal structure of EzrA, which rather unexpectedly adopts the same fold as the spectrin family of cytoskeletal proteins. Spectrins have a simple, but unique, repetitive domain structure, comprising between 4 and 20 of the repeats in a linear array. EzrA has five repeats, arranged in a linear array that curves to form a semi-circle. Spectrins link the actin/tubulin cytoskeleton to the membrane by interacting with membrane-embedded proteins; EzrA interacts with both FtsA (actin) and FtsZ (tubulin) as well as the membrane-embedded proteins, GpsB and major peptidoglycan synthases. Hence EzrA acts in the same way as the spectrins. This proposal seeks to understand at a molecular level how these interactions are driven, what the key amino acids are, and what the consequences are on cell division if the essential interacting interfaces are mutated.

This is a basic biology project, with no immediate impact on non-academic beneficiaries. Cell division is a fundamental process, essential to all domains of life. A complete knowledge of cell division is an essential requirement of understanding the healthy cell. Though we tend to associate the healthy cell with the self, our relationship with bacteria is a complex one, and it is now emerging that a healthy microbiome is a key indicator of the health of the individual. Furthermore, though beyond the scope of this project, the march of multidrug resistant strains of pathogenic bacteria, and the failure of novel antibiotics to make the market place, is a current area of concern for public health. A greater depth of understanding of cell division, and the appropriate identification of new target proteins and the small molecules that inhibit these proteins' functions, is therefore of great importance and likely to lead to significant impact on the whole of society. Whilst this project is not designed to probe EzrA's essentiality to Staphylococcus aureus, any future commercially exploitable results will enter the market place after the protection of our IP through the University of Newcastle's Business Development Unit. These connections are with UK-based companies, hence the results of this project is likely to benefit directly the UK economy.