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Piggy-backing the bacterial chromosome: positioning of protein complexes by chromosome segregation

Reference	BB/L002507/1
Principal Investigator / Supervisor	Professor Judith Armitage
Co-Investigators / Co-Supervisors
Institution	University of Oxford
Department	Biochemistry
Funding type	Research
Value (£)	346,066
Status	Completed
Type	Research Grant
Start date	01/10/2013
End date	30/09/2016
Duration	36 months

Abstract

Most bacterial species use a chromosome associated ATPase to segregate large plasmids. The ParA ATPase protein loosely coats the chromosome as dimers or filaments, depolymerising on interaction with a ParB protein carrying the plasmid "cargo". In a mechanism as yet unclear, this keeps the cargo associated with the chromosome. When the chromosome and plasmids duplicate the plasmids segregate with the segregating chromosomes and each daughter inherits the correct number of plasmids. It has recently been shown that a related system may segregate protein complexes that need to be in functional complexes. The best characterised is involved in segregating the large cytoplasmic chemosensory complex of Rhodobacter sphaeroides. In this case a ParA-like protein, PpfA, encoded in the operon encoding the chemosensory proteins, loosely associates, non-specifically, with the chromosome. A chemoreceptor, TlpT, both brings all the thousands of copies of 8+ different chemosensory proteins together into a cluster and activates the ATPase activity of PpfA: acting like ParB to both carry the cargo and drive the segregation activity of the ParA-like ATPase. Using fluorescence microscopy, PALM and FRET, we will characterise the behaviour of PpfA on the chromosome surface and the interactions between PpfA and TlpT. Using microfluidics and chromosome release plus in vivo and in vitro protein chemistry we will characterise the organisation of the cluster on the chromosome surface and the dynamics of the proteins within the cluster. We will characterise the mechanisms involved in forming a new cluster, and segregating the cluster as the chromosomes segregate plus identify the minimal system able to segregate. This minimal system will be harnessed to move unrelated proteins between cells in defined ratios for use in forming and segregating "metabolic factories" at local high concentrations between cells, providing part of the developing bacterial "chassis" for use in synthetic biology.

Summary

When a cell divides, in general it produces a reasonable replica of itself. As the cell grows it makes new proteins, so that, before division it is reasonable to assume it has twice as many proteins as the initial cell. When that cell divides each daughter needs a chromosome to make those proteins, but are the proteins just a free soup that gets randomly divided between the daughter cells? That may be the case for some proteins, but other proteins need to be divided with partner proteins in the right ratio to work. This can be done by making tight, stable complexes that get divided together, but other groups of proteins need mechanisms that ensure each daughter cell has the correct proteins in the correct ratio. Membranes have long been know to be provide a framework for allowing proteins to come together, organising receptor proteins that respond to changes in the external world into interacting protein complexes. What about proteins that are soluble and do not respond to the external environment-how to they organise and how do cells ensure daughter cells inherit the right number of complexes. It has recently been realised that in bacterial cells some protein complexes can "piggy-back" on the duplicated and segregating chromosomes, with a protein loosely covering the chromosome surface and also interacting with a complex of proteins. As the chromosome segregates this protein carries this large protein "cargo" to the daughter cell. The protein which loosely associates with the chromosome is related to proteins known to also ensure daughter cells inherit the right genetic material and it is now clear that bacteria use this system to segregate a wide range of protein complexes needed in the correct ratio to function properly. As this is a common system across bacteria, it could be harnessed to organise other protein complexes between bacteria. In many metabolic pathways efficient rates are achieved when proteins are closely associate ensuring local high concentrationsand fast substrate transfer. If these pathways are expressed in alien systems the proteins are often diffuse and disorganised, resulting in inefficient rates of activity. In addition many proteins function with partner proteins and to understand how they work ideally their structures need to be characterised when bound, but usually the proteins are purified individually and mixed or when expressed individually they are insoluble. It is possible we can use this natural system for segregating protein complexes to keep other proteins in functional complexes and at high local concentrations. Using this reduced system we could either use the system in synthetic biology for allowing a daughter cells to segregate proteins in functional but high ratios for a pathway to efficiently produce a desired product or it will allow expression of protein partners in an environment that will allow their isolation and structural analysis. To be able to harness this newly identified mechanism we need to understand the essential components holding the proteins together as complexes, causing the protein complexes to duplicate and the association with the chromosome on the chromosome associated protein.

Impact Summary

Who will benefit from the research: The immediate impact will be researchers in bacteriology who are interested in the mechanisms involved in chromosome organisation and in formation and segregation of critical protein complexes between daughter cells. As the work progresses it will be of interest to researchers working in the area of nanotechnology, systems biology and synthetic biology as it will provide a novel framework for developing chromosomes and associated proteins as a mechanism for regulation of positioing of metabolic "factories". The research will push the development and use of microfluidic devices and in vivo cell imaging and the development of spatio-temporal modelling at a systems level. How will they benefit: The project will combine molecular, imaging and nanotechnological approaches to describe the mechanisms underpinning protein dynamics and cluster formation in living cells and develop new technologies for investigation the dynamics of the proteins associated with chromosome surfaces in vivo and in vitro. It will identify the commonality within systems driving chromosome, plasmid and protein segregation and identify whether there is a core set of protein domains that will both help define a system and enable it to be used to organise a wide range of "cargoes" at cell lengths using any segregating chromosome system-allowing spacing at equal intervals of interacting proteins at high local densities. There is an increasing interest in the use of the basic bacterial "chassis" as a vehicle for organising a wide range of synthetic processes. Many metabolic pathways operate more efficiently is they are brought together in a specific ratio in high concentration to provide "metabolic factories". These usually require the membrane as an organising structure, but the bacterial membrane has a limited area and overexpression of membrane proteins can be toxic. The system under study here operates across bacterial species and uses the chromsome surface to organis and segregate proteins in the correct ration, and can segregate 1000s of proteins and is related to systems shown to organise systems as unrelated as carboxysomes and pathogenicity complexes. Understanding the core mechanism involved will allow the system to be exploited for small metabolic pathways that may, for example, produce toxic or short lived intermediates. In the medium to long term this may therefore be exploited in the developing synthetic biology community to produce end products that are difficult to synthesise chemical, but are also difficult to make using standard biotechnology because of intermediates unstable in solution.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Microbiology, Synthetic Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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