How does a chimeric partition machine mediate chromosome segregation in Archaea?

Archaea evolved as the third 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 37 years ago represented a major biological 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). Initially isolated from extreme ecosystems, archaea are now known to be ubiquitous, constituting a considerable fraction of the biosphere. For example, it has been reported that the world ocean alone contains approximately 1.3 x 10 to the 28 archaeal cells: this is an enormous number. To provide a comparison, the estimated number of grains of sand on all the beaches on earth is 7.5 x 10 to the 18, a quantity still much smaller as compared with that of marine archaeal cells. Their ubiquity and abundance make them key players in regulating global biogeochemical cycles on Earth. From a functional and mechanistic standpoint, archaea are a mosaic of tesserae from bacteria and eukaryotes, but they are also characterized by unique molecular features like methane production.
Thermophilic 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 basic studies on life pushed to extremes. The heat resistant molecules found in thermophilic archaea (for example proteins and lipidic chains) have revealed to us that the boundaries of life as we know it can be pushed much further than previously anticipated. Their ability to grow in extreme environments where no other terrestrial organism can survive has also rejuvenated hopes of discovering extraterrestrial life on inhospitable planets.
Despite the significant progress made in decoding molecular mechanisms in these organisms in the last three decades, to date little information is available on the fundamental process of chromosome 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 genetic material is first duplicated, then separated and equally distributed into the two daughter cells. We intend to dissect this process in the thermophilic archaeon Sulfolobus solfataricus, whose genome encodes for two proteins, SegA and SegB, which interact to form a simple chromosome segregation machine.
The proposed project intends to discover the mechanisms adopted by the SegAB complex to mediate the separation and equi-distribution of chromosomes in S. solfataricus at cell division. We will shed light on the localization of these proteins in the cell by fusing them to a heat stable fluorescent protein and using conventional microscopy and a novel imaging technique, called super resolution microscopy. This approach will allow us to acquire a high-resolution picture of the structures formed by SegA and SegB in the cell. We also wish to investigate the interaction of each of the proteins with DNA to map their binding sites on the chromosome and to understand how these associations result in chromosome segregation. Another aim of the work is to identify other proteins that interact with the SegAB complex inside the cell: two different screening strategies will be expolited, one looking for genes and the other looking for proteins of potential partners. In addition, we want to determine the three-dimensional structure of SegA and SegB. The multiple pieces of the jigsaw deriving from the various investigations will be combined to generate a detailed picture of chromosome segregation in S. solfataricus

Chromosome segregation is a fundamental biological process in all organisms. It requires the concerted action of dedicated proteins and its coordination with cellular events, such as DNA replication and cell division, is crucial to maintain euploidy. The molecular mechanisms promoting chromosome partitioning in eukaryotes are well characterized. The key players behind genome segregation in prokaryotes are not fully elucidated. However, considerable progress has been made to decipher this process in bacteria in the last two decades.

In contrast, the molecular mechanisms and factors underpinning chromosome segregation in archaea, the third domain of life, are entirely underexplored, a terra incognita awaiting investigation. We have shown that the archaeon Sulfolobus solfataricus harbours a hybrid segregation machine consisting of two interacting proteins, SegA and SegB, that play a key role in chromosome segregation in this organism. SegA is an ortholog of bacterial Walker-type ParA proteins, whereas SegB is an archaea-specific factor that works as a DNA anchor binding palindromic motifs. SegA self-assembles into higher order structures and this property is synergized by SegB. The picture emerging from our findings indicates that the SegAB complex fulfils a crucial function in chromosome segregation and is the prototype of a DNA partition system widespread across archaea.

The question that we intend to address here is: how does this hybrid partition machine mediate chromosome segregation? Multidisciplinary approaches will be adopted to gain a mechanistic understanding of SegAB modus operandi. The interplay between proteins and chromosome will be probed at molecular and cellular levels by using genetic tools in parallel to novel technologies such as super resolution microscopy. The investigations will provide exciting new perspectives on chromosome biology that will inform and broaden mechanisms and principles established for the other two domains of life.

The project will have repercussions in a number of impact areas reported below. Different mechanisms will be used to achieve the impact objectives.

SCIENTIFIC IMPACT
The results generated by the proposed investigations will lead to 2 - 3 publications in high-impact factor journals within a time frame of 2 to 4 years from the start of the project. The findings will be published in open access journals or journals that provide the open access option, so that they will be available to widest scientific audience possible as soon as the papers are published.
The team (PI and postdoctoral RA) will communicate the research findings of this project at national and international conferences for which funding has been requested.
The genetic and microscopy experiments outlined in the proposal will lead to the development of novel and much needed tools that the archaea community would benefit from.

SOCIETAL IMPACT - TRAINING
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 his/her next career move. Working in the departmental state-of-the-art Technology Facilities (Genomics, Proteomics and Imaging labs) will allow the RA to acquire invaluable training in the use of sophisticated instruments and unparalleled support in data analysis. The research technician will receive significant training in protein purification and molecular cloning 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 applicant is involved in UCAS and Open days to recruit new students and thus interacts with young people and their families. The team working on the proposed project (PI, RA and RT) will set up a stall entitled 'Archaea: Life at the edge of Survival' in the atrium of the Department and will engage the visitors in discussions about archaea and the specific project.
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 also at this festival in 2016 and 2017.
We will set up a webpage dedicated to the proposed project and will update it systematically with news concerning results, achievements and the life of the 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 COLLABORATION
For the proposed project we will collaborate with Dr Maria Schumacher (Duke University) to solve the structures of SegA and SegB proteins. This collaboration fits squarely in the BBSRC strategic priority of 'Increased international collaboration'.