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Professor Daniela Barillà

Professor of Prokaryotic Genetics


PURE Staff link York Research Database

The precise distribution of newly replicated genomes to progeny cells is crucial for stable transmission of genetic information. Bacteria and Archaea employ dedicated DNA segregation factors that drive and deliver low copy number plasmids and chromosomes to specific subcellular locations.

Segregation mechanisms of a multidrug resistance plasmid

One of the model systems under investigation is the multidrug resistance plasmid TP228 that replicates at low copy number in Escherichia coli. This mobile genetic element specifies resistance to a range of antibiotics, including tetracycline, streptomycin and sulphonamides. The plasmid harbours a partition cassette encoding two proteins that are crucial for plasmid inheritance: ParF, a ParA Walker-type ATPase, and ParG, a site-specific DNA-binding protein. We have employed multidisciplinary approaches, spanning from genetics to biochemistry, from super resolution microscopy to biophysics, to investigate the role and interactions of these proteins in mediating plasmid segregation.

Figure 1. Structure of the ParF protein bound to the ATP analogue AMPPCP (cyan), one monomer is shown in blue-green and the other in red-orange.

ParF Structure

We have solved the three-dimensional structure of ParG and ParF. Crystal structures of the ParF in different nucleotide-bound states suggest a mechanism underlying higher-order structures formation (Fig. 1). 

Figure 2. A 3D ParF meshwork permeates the nucleoid.3D structural illumination microscopy images of an E. coli cell. ParF is fused to GFP Emerald and the ParG to mCherry. The nucleoid is stained with DAPI.

Recent microscopy work from our group has shown that ParF assembles into a dynamic three-dimensional meshwork that permeates the nucleoid and periodically shuttles between the nucleoid poles(Fig. 2). We have proposed a Venus flytrap model in which the ParF lattice captures plasmid-ParB analogue complexes within the nucleoid volume and positions them through assembly/disassembly cycles caused by ParF ATP binding and hydrolysis respectively [NAR 2017, 45: 3158-3171].

Discovering the mechanisms whereby multidrug resistance plasmids are inherited will ultimately help us to develop novel therapeutic agents to combat bacterial infections.

Genome segregation in thermophilic Archaea

Archaea are fascinating objects of investigation as they can thrive in very harsh environments thanks to molecular adaptations to a life pushed to extremes. Genome segregation in the archaea domain is virtually an uncharted territory. Two strands of research are currently being pursued, one focusing on mechanisms responsible for chromosome segregation in Sulfolobus solfataricus [Proc Natl Acad Sci USA 2012, 109: 3754-3759] and the other investigating the segregation of a low copy number plasmid in a different Sulfolobus species isolated from acidic hot springs in the island of Hokkaido, Japan. The toolkit for the stable inheritance of this plasmid is a unique three-component machine borrowing building blocks from bacteria and eukarya, including a CenpA-like histone. One of the proteins, AspA, binds to the centromere site, spreading on the DNA and building a superhelix template onto which the other two factors assemble to mediate plasmid segregation [Science 2015, 349: 1120-1124].

 Figure 1. Structure of the ParF protein bound to the ATP analogue AMPPCP (cyan), one monomer is shown in blue-green and the other in red-orange.

 

Contact details

Professor Daniela Barillà
Professor of Prokaryotic Genetics
Department of Biology
University of York
Heslington
York
YO10 5DD

Tel: 01904 328715

https://barillalabyork.weebly.com/