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

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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