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

Lead Research Organisation: University of York
Department Name: Biology

Abstract

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

Technical Summary

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.

Planned Impact

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'.

Publications

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Barilla` D (2017) Genome segregation in heat-loving archaea in Microbiology Today

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Barillà D (2016) Driving Apart and Segregating Genomes in Archaea. in Trends in microbiology

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Kamada K (2018) Combing Chromosomal DNA Mediated by the SMC Complex: Structure and Mechanisms. in BioEssays : news and reviews in molecular, cellular and developmental biology

 
Description This project focused on a heat-loving microbe called Sulfolobus that thrives in very hot environments such as hot springs. Members of the genus Sulfolobus are fascinating objects of investigation, as these microbes live lives pushed to extremes where no other terrestrial organism could survive. This lifestyle 'on the edge of survival' is made possible by sophisticated adaptations in the molecular building blocks of the cells. As life might have originated in extremely hot environment like hydrothermal vents, studying microbes living in extreme ecological niches might shed light on the origin of life. Our investigations focused on an essential biological process, known as chromosome segregation. Every cell has one or more molecules of DNA that harbour a code for all the proteins that are produced by the cell. These DNA molecules can be either linear, such as those in our cells, or circular, such as those found in Sulfolobus cells. During the course of this project we have studied how chromosomes are partitioned into two daughter cells, when a mother cell divides. We have identified two proteins that are involved in this process. The proteins have been named SegA and SegB (for chromosome segregation). We have discovered that the proteins interact with one another, working in a synergistic fashion to mediate chromosome transfer from mother to daughter cell at the time of cell division. When one of the proteins is absent or defective, the process is affected, resulting in cells with no chromosome. The use of microscopy techniques has allowed us to define the cellular localization of SegA and SegB as well as their interaction with other cellular components. Localization patterns and network of interactions have provided insights into the mechanism by which this simple system operates in Sulfolobus cells.
Exploitation Route The project has provided novel insights into how chromosome segregation operates in the archaea domain of life.
Sectors Education,Pharmaceuticals and Medical Biotechnology

 
Description We have participated in a public engagement event that was part of the York Festival of Ideas 2016 and 2017. This event has provided us with the opportunity to communicate the aim and results of our project to the general public. My team took part in the Discovery Zone activity, part of the York Festival of Ideas, in 2018. We had a stall describing the importance of Archaea. Over 2000 people visited our stall in 2016, 2017 and 2018.
First Year Of Impact 2016
Sector Education
Impact Types Cultural,Societal

 
Title Generation of a Sulfolobus solfataricus genomic library 
Description Generation of a S. solfataricus genomic library to be used for a genetic screen 
Type Of Material Biological samples 
Provided To Others? No  
Impact The genomic library will allow to identify proteins interaction with the chromosome segregation system that we work on. 
 
Title Generation of antibodies against SegA and SegB proteins 
Description These antibodies are tools useful to investigate proteins localization in the cell. 
Type Of Material Antibody 
Year Produced 2016 
Provided To Others? No  
Impact These antibodies have allowed us to investigate the localization of the proteins under study in the cells and these experiments have shed light on how the system works. 
 
Description ChIP-seq 
Organisation John Innes Centre
Department Molecular Microbiology
Country United Kingdom 
Sector Private 
PI Contribution The research associate funded by the grant visited the laboratory of Dr Tung Le, John Innes Centre, to learn chromatin immunoprecipitation associate with deep sequencing.
Collaborator Contribution Our collaborator, Dr Tung Le, is an expert in ChIP-seq. This collaboration allowed us to master this cutting-edge technique.
Impact No outputs as yet, a manuscript is in preparation.
Start Year 2017
 
Description Science out of the Lab - York Festival of Ideas 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We had a stall in the city centre and engaged children, young and adult people by telling them about archaea and getting them involved in interactive activities/experiments. Over 2000 people visited during two days.
Year(s) Of Engagement Activity 2017
 
Description Talk at EMBO Meeting on Archaea, Vienna, September 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I gave a talk about the progress of our work on chromosome segregation in Sulfolobus solfataricus.
Year(s) Of Engagement Activity 2018
 
Description Talk at EMBO Meeting on DNA replication, chromosome segregation and fate decisions, Kyllini, Greece 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I gave an invited talk on our work on chromosome segregation in Sulfolobus solfataricus. The talk generated a substantial discussion with the audience.
Year(s) Of Engagement Activity 2018
 
Description York Festival of Ideas - Science out of the lab 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact My group and myself had stalls in the city centre and interacted with the general public, from young children to teenagers to adults. We introduced people to the concept of Archaea as a different domain of life by using postcards, T-shirts and easy lab activities such as plating cultures or smelling plates.
Year(s) Of Engagement Activity 2016
URL http://yorkfestivalofideas.com/2016/