A novel DNA segregation model system from Archaea revealing bacterial and eukaryotic linkages

Archaea evolved as a domain of life billions of years ago, but they are a relatively recent addition to the map of the universal tree of living organisms. Their discovery 40 years ago represented a major milestone. Archaea are unicellular organisms that populate our planet together with bacteria and eukaryotes. Both bacteria and archaea are prokaryotes, i.e. their genetic material is not wrapped by a membrane into a separate compartment, called nucleus, which is instead a hallmark of eukaryotes (baker yeast, fungi, plants, animals and humans to mention some). Archaea are known to be ubiquitous, constituting a considerable fraction of the biosphere. Their ubiquity and abundance make them key players in regulating biogeochemical cycles on Earth. From a functional and mechanistic standpoint, archaea are a mosaic of features from bacteria and eukaryotes, but they are also characterized by unique features like methane production.
Heat-loving archaea are super microbes thriving at 80 degrees C and higher temperatures in hot springs, volcanoes, deep sea vents and exhibiting unusual properties, which make these organisms valuable for the development of novel biotechnological applications, but also extremely interesting for studies on life pushed to extremes. Their ability to grow in extreme environments where no other terrestrial organism can survive has also rejuvenated hopes of discovering extraterrestrial life.
Despite the significant progress made in decoding molecular mechanisms in these organisms in the last four decades, to date little information is available on the fundamental process of DNA segregation in archaea and the subject remains a black box awaiting investigation. Genome segregation is a crucial stage of the life cycle of every cell: the DNA is first duplicated, then separated and equally distributed into the two daughter cells. We intend to study this process in a strain of the heat-loving archaeon Sulfolobus isolated from an acidic hot spring in Japan. This microbe contains a large and a small ring of DNA. The large is called chromosome and the small is designated as plasmid. We have recently investigated three proteins that are encoded by the plasmid, pNOB8, and solved their three-dimensional structures. These proteins assemble into a nanomachine that drives duplicated sister plasmids apart, so that each daughter cell receives the same copy number.
We intend to introduce changes in the DNA of the host Sulfolobus strain, so that mutations can be introduced in the genes encoding the proteins to show that these factors are essential for the inheritance of the plasmid. One of the proteins responsible for the inheritance of pNOB8 is AspA. It binds strongly to a special site on the plasmid that acts as a docking site and then associates to adjacent regions spreading on the DNA and forming a helix. We want to investigate whether the plamid contains multiple docking sites for AspA and the process through which this protein stretches on the DNA. The other two proteins of the plasmid segregation nanomachine are ParB and ParA. ParB is an adaptor sitting between AspA and ParA in the complex. Adaptors need to be pliable and ParB is indeed flexible: the protein consists of two domains connected by a flexible linker. One of the ParB domains has a bacterial flavour, whereas the other looks like a eukaryotic protein. We suspect that the ParB region that binds ParA is the flexible linker and we want to test this hypothesis. We intend to investigate if other proteins in Sulfolobus associate with the eukaryotic domain of ParB, modifying and regulating it. Finally, we are eager to gain a snapshot of these proteins in Sulfolobus cells. We are going to use microscopes that will provide high-resolution images of the protein complex and will tell us where it localizes relative to the DNA mass of the chromosome. This analysis will allow to identify patterns that will shed light on the life of the pNOB8 DNA segregation nanomachine.

Genome segregation is a fundamental biological process that ensures survival in all organisms. The events mediating chromosome segregation in eukaryotes are well understood. The mechanisms that drive DNA segregation in bacteria are not fully elucidated, but significant progress has been made in the past two decades.

In contrast genome segregation in archaea, the third domain of life, remains a terra vastly incognita despite considerable achievements in other areas of archaeal biology. We have investigated a novel DNA segregation system from archaea and structural studies have disclosed bacterial and eukaryotic linkages. This DNA partition machine consists of a Walker-type ParA, a ParB adaptor and a unique centromere-binding factor, AspA. The AspA protein spreads from its binding site on the DNA, generating a helical docking platform onto which the N-terminal domain of ParB assembles fitting in a lock-and-key fashion into the AspA-DNA structure. The ParB C-terminus, which binds non-specific DNA, shows structural similarity to the CenpA histone variant, which demarcates centromeres and is vital for kinetochore assembly in eukaryotic cells. ParA is recruited onto the complex via interaction with ParB. This multi-protein structure merges bacterial and eukaryotic features suggesting the possible conservation of DNA segregation principles across the domains of life.

We are now in an ideal position to discover key principles underpinning the mode of action of the AspA-ParBA system and to open up fresh perspectives on genome segregation in archaea. We intend to gain an in vivo snapshot of how this complex acts in the cell by performing genetic and high-resolution microscopy investigations. Furthermore, role and interplay of the proteins will be studied exploiting multidisciplinary approaches. The findings from this project will provide a mechanistic understanding of archaeal genome segregation and will inform principles established for the other two domains of life.

This project involves a mix of experimental strategies that will provide the postdoctoral RA with an excellent and versatile portfolio of skills and expertise, which will make her/him very marketable as researcher in both academia and industry for the next career move. Working in the departmental state-of-the-art Technology Facility (Molecular Interactions, Proteomics and Imaging Labs) will allow the RA and RT to acquire invaluable training in the use of sophisticated instruments and unparalleled support in data analysis. The research technician will receive significant training in genetics, molecular biology and protein purification and will work closely with RA and PI, benefitting from their expertise.
Attending national and international conferences will provide opportunities for the RA to develop presentation/communication skills and to forge links with colleagues and organizations working in the same field.
The RA will have the opportunity to supervise undergraduate students carrying out their final year project in our laboratory. This experience will provide the RA with valuable supervision skills.

SOCIETAL IMPACT - PUBLIC ENGAGEMENT
We recognise the importance of divulging the findings of this research project to the greater public and we will achieve this aim through a number of mechanisms.

The University of York organizes a Festival of Ideas, whose theme changes every year. Science is an important aspect of this event and thus we will feature our stand on archaea at this festival in 2018, 2019 and 2020. The team (PI, RA and RT) will set up a stall entitled 'Archaea: Life at the edge of Survival' and will engage the visitors in discussions about archaea and the specific project. The event takes place in York city centre and people of any age drop in. As most of the people with no scientific training are unlikely to have heard about archaea, the team will be creative and will use posters, leaflets, T-shirts, mugs, archaea stickers, microscopy high resolution images and 3D-printed models of the protein structures (funds have been requested for these items). We will engage children inviting them to perform simple experiments using the brilliant Bento Lab, a portable min-lab that includes a centrifuge, a gel electrophoresis unit and a PCR thermocycler. This way we hope to capture the public's attention and make our results accessible to a wide audience.
We will also take part in the Royal Society Summer Science Exhibition in London in 2020. By then, we will have sufficient results and material to organize multiple activities for this week-long exhibition.

In addition, we will organize visits to local primary schools and prepare activities suitable for young children, making the most of the portable Bento Lab.

We intend to make press releases about publications deriving from the project and write lay audience articles in popular science magazines.

BROADER IMPACT - INTERNATIONAL PARTNERSHIP
For the proposed project we will collaborate with Professor Qunxin She (University of Copenhagen) to set up a genetic system for Sulfolobus NOB8H2. This collaboration fits squarely in the BBSRC strategic priority of 'International Partnership'.