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Role of a ParA homologue in segregating the chemosensory protein cluster of Rhodobacter sphaeroides

Reference	BB/C511880/1
Principal Investigator / Supervisor	Professor Judith Armitage
Co-Investigators / Co-Supervisors
Institution	University of Oxford
Department	Biochemistry
Funding type	Research
Value (£)	229,609
Status	Completed
Type	Research Grant
Start date	01/03/2005
End date	29/02/2008
Duration	36 months

Abstract

Bacterial chemotaxis is essential for colonisation of a wide range of habitats. It has also provided a paradigm for the two component sensory systems involved in regulating expression of a very wide range of pathways. However, the E. coli pathway which has been the model for many years has been found to be much simpler than the pathways in most other bacterial species with sequenced genomes. The majority of species have multiple components of the chemosensory pathway and we have identified that, in R. sphaeroides, these different pathways do not cross talk in vivo (although they do in vitro) because the components of the pathways are physically separate in the cell. In cephalexin induced filaments the chemosensory cluster segregate evenly down the filament, indicating control of both targeting and segregation. Data from a number of groups have identified a family of proteins responsible for segregating plasmids and chromosomes between dividing cells, ensuring each daughter cell has the same DNA complement after septation. The operon which encodes the proteins which form the cytoplasmic chemosensory cluster in R. sphaeroides also encodes a protein with homology to the Walker-type parA ATPase involved in plasmid segregation. Deletion of this homologue, PpfA resulted in a strain with reduced responses and when cluster segregation was examined we found only one cluster per cell before septation, rather than the usually segregated two per daughter cell. Even more dramatically, cephalexin treated cells had only one cluster per filament. This suggests that PpfA is involved in both regulation of expression of the chemosensory operon, possibly linked to septation events, and in controlling segregation at cell division. PpfA must interact with the upstream promoter of CheOp3 or with a repressor expression in the absence of PpfA and linked to a septation event. We will characterise the control of expression by PpfA using lacZ fusions, retardation and DNA footprinting using wtand site directed mutant protein. It is likely from the phenotype that PpfA is the anti-repressor however, and to identify the protein with which it interacts we will use yeast two hybrid, immunoprecipitation and second site suppressor studies, isolating PpfA mutants with restored wt chemotaxis. The data show that the cytoplasmic cluster is regulated both transcriptionally and physically in the cell, segregating at almost even intervals along an induced filament. This suggest that interacting proteins involved in signalling may need to be present in stoichiometic amounts and that this needs to be highly regulated. We will identify the proteins responsible for controlling the segregation of the chemosensory clusters. This indicates a cytoskeleton-like system involved in segregating proteins to ensure accurate partitioning. Identification and mutagenesis of this protein will allow us to eventually identify which other pathways might be similarly regulated. Again this will use yeast two hybrid and immunoprecipitation. PpfA is not responsible for the formation of the clusters, only the expression and the segregation and possibly the positioning in the cell. One of the four cytoplasmic chemoreceptors encoded by R. sphaeroides is essential for both chemotaxis and cluster formation, TlpT. This is the only cytoplasmic receptor with a HAMP domain, identified as essential for dimer formation and protein:protein interaction in many membrane spanning proteins. We will identify whether TlpT and SlpA interact in vitro by site specific mutagenesis and BiaCore studies. If they interact in vitro, in vivo interaction will be measured by FRET analysis. The extent of fluorescence at the poles and in the clusters increases with time. To identify whether new protein is targeted to new poles/clusters preferentially over old poles/clusters or whether all clusters are equal and the timing of new cluster formation, we will use FRAP and follow the development of fluorescence.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	X – not assigned to a current Research Topic
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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