Probing DNA segregation in archaea: molecular dissection of an atypical tricistronic partition system from Sulfolobus
Lead Research Organisation:
University of York
Department Name: Biology
Abstract
The process of genome segregation is a fundamental stage of the life cycle of each cell: the genetic content is first duplicated, then separated and equally distributed into the two daughter cells. The mechanism whereby the genetic material (that is organized into linear DNA chromosomes) is separated in cells of higher organisms (like plants, animals, humans) has been extensively studied and is well understood. In these cells the molecular machine responsible for chromosome segregation is known as 'mitotic spindle': it consists of cables, called microtubules, which are anchored to chromosomes at a specific site, known as 'centromere'. The microtubules pull sister chromosomes to opposite poles of the spindle, before the cell divides. In bacteria the picture is more elusive. The genetic patrimony of bacteria consists of a single circular (more rarely linear) chromosome and sometimes smaller circles of DNA called plasmids. Historically, it was presumed that segregation of bacterial chromosomes and plasmids was a passive process, not requiring a dedicated apparatus and perhaps involving attachment of the newly-replicated genetic elements to the growing cell wall. However, in recent years evidence has been provided pointing to the existence of an active mechanism responsible for the segregation of chromosomes and plasmids, requiring the participation of dedicated factors. In bacteria, the most well-characterized DNA segregation systems are those specified by plasmids, which are present in the cell in low numbers. These plasmids harbour their own survival kit, a segregation cassette consisting of two genes, often termed parA and parB, and a centromere-like site. This cassette ensures an accurate segregation of the plasmids from one generation to the next at cell division. The molecular mechanisms underlying this process have not been thoroughly elucidated as yet; however, recent discoveries hint at the existence of mitotic spindle-like machineries. Archaea are the third domain of life: their discovery in 1977 represented a major biological milestone. They were initially identified as a different group of organisms on the basis of sequences contained in their RNA or ribonucleic acid. Further characterization of their physiology, biochemistry and genetics has provided unequivocal evidence that they are a distinct group of organisms (different from bacteria and from higher multicellular organisms). The first archaea to be studied were all from extreme environments, but they are now known to thrive in most of the earth's ecological niches and they constitute ~20% of the biosphere. Hyperthermophilic archaea grow at 80 C and above and exhibit unusual properties, which make these organisms a valuable resource for the development of novel biotechnological processes. Industrial applications include the production of archaea-derived enzymes, which are stable at high temperature, cellulose degrading enzymes, the use of their membranes as delivery systems for drugs and genes. Despite numerous studies on fundamental biological processes in archaea, to date no information is available on the mechanism of genome segregation in these organisms. We intend to analyze this process in an archaeon called Sulfolobus NOB8H2, which has been isolated from hot springs in the island of Hokkaido, Japan. This archaeon contains a plasmid, pNOB8, which harbours a putative DNA segregation cassette comprising three genes (designated as orf44, parB, parA). The project here proposed will focus on the characterization of the factors encoded by the genes above, their function and respective role in pNOB8 partitioning at cell division and their dynamic interactions. We also intend to identify the centromere of pNOB8 and dissect the interactions between this site and ParB, ParA and perhaps Orf44. Furthermore, investigations will be conducted to probe whether the ParA protein assembles into cable-like structures as observed for some bacterial ParA counterparts.
Technical Summary
Genome segregation is a fundamental process in all organisms: it requires the concerted action of dedicated proteins and its timing and coordination with other cellular events, like DNA replication and cell division, is crucial to maintain euploidy. The molecular mechanisms promoting accurate chromosome partitioning in eukaryotes are well characterized. Bacterial DNA segregation has been explored mainly in model organisms like Escherichia coli, Bacillus subtilis and Caulobacter crescentus. Biochemical, structural and cell biology investigations have shed light on a growing number of cytoskeletal elements responsible for the segregation of chromosome and plamids in bacteria. In contrast with eukarya and bacteria, to date the mechanisms and dynamics of genome partitioning are entirely underexplored in archaea. The atypical orf44-parB-parA module of Sulfolobus NOB8H2 provides a particularly good platform to initiate a survey of DNA segregation in archaea.The three genes partially overlap, which suggests that they might be part of a single transcriptional unit. The products encoded by parB and parA genes exhibit homology, respectively, to the ParB and ParA superfamilies of bacterial DNA partition proteins, whereas Orf44 shares similarity with bacterial repressors of the ArsR family. The proteins encoded by this putative partition cassette are easily purified and amenable to molecular dissection. By using complementary approaches, we intend to elucidate the molecular mechanism underlying the segregational stability of pNOB8. The respective role played by ParB, ParA and Orf44 in plasmid partitioning at cell division and their interaction dynamics will be investigated. Parallel studies will focus on the identification of the centromere of pNOB8. A final objective is to investigate whether ParA is a motor protein capable of polymerization. These studies will provide valuable new perspectives on DNA segregation in an important and widespread group of organisms.
People |
ORCID iD |
Daniela Barillà (Principal Investigator) |
Publications
Hayes F
(2010)
Bacterial Chromatin
Kalliomaa-Sanford AK
(2012)
Chromosome segregation in Archaea mediated by a hybrid DNA partition machine.
in Proceedings of the National Academy of Sciences of the United States of America
Barillà D
(2016)
Driving Apart and Segregating Genomes in Archaea.
in Trends in microbiology
Caccamo M
(2020)
Genome Segregation by the Venus Flytrap Mechanism: Probing the Interaction Between the ParF ATPase and the ParG Centromere Binding Protein.
in Frontiers in molecular biosciences
Barillà D
(2010)
One-way ticket to the cell pole: plasmid transport by the prokaryotic tubulin homolog TubZ.
in Proceedings of the National Academy of Sciences of the United States of America
Schumacher MA
(2015)
Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages.
in Science (New York, N.Y.)
Description | Factors involved in DNA segregation in archaea, the third domain of life. We have solved the three-dimensional structures of the three proteins of this DNA segregation system. |
Exploitation Route | I guess they contribute to broaden the knowledge on archaea and open the path to exploitation of these organisms. |
Sectors | Education Environment Pharmaceuticals and Medical Biotechnology |
URL | http://www.sciencemag.org/content/349/6252/1120 |
Description | The findings have been included in peer reviewed publications. They have also been reported in a press release. |
First Year Of Impact | 2012 |
Sector | Education,Environment |
Impact Types | Cultural Societal |
Description | Responsive mode research grant |
Amount | £527,536 (GBP) |
Funding ID | BB/R006369/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 05/2018 |
End | 05/2021 |
Title | Clones for the production of recombinant archaeal proteins in E. coli |
Description | We generated various clones expressing archaeal DNA segregation genes. |
Type Of Material | Biological samples |
Provided To Others? | No |
Impact | Using these clones we were able to purify proteins whose structures have now been solved. |
Description | Maria Schumacher - Structure of Sulfolobus DNA segregation proteins |
Organisation | Duke University Medical Centre |
Country | United States |
Sector | Academic/University |
PI Contribution | We have provided recombinant plasmids and purified proteins. In addition, we have contributed expertise in DNA-protein interactions and protein-protein interactions. |
Collaborator Contribution | Maria has solved numerous structures of proteins dissected with BBSRC funding. |
Impact | A number of manuscripts are in preparation. |
Start Year | 2009 |
Description | Sonja V Albers collaboration |
Organisation | Max Planck Society |
Department | Max Planck Institute for Terrestrial Microbiology |
Country | Germany |
Sector | Academic/University |
PI Contribution | We developed a collaboration that has resulted in a joint high profile publication on which the collaborators are co-authors. |
Collaborator Contribution | We have acquired tools and skills in Sulfolobus genetics. |
Impact | Generation of Sulfolobus knockout strains; one publication in a high profile journal. |
Start Year | 2008 |
Description | International Plasmid Biology Conference 2010. Bariloche, Argentina, 2010. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | One postdoc presented a poster on our multidrug resistance plasmid segregation project (MRC). Another postdoc gave a talk about our archaea project (BBRSC). Promotion of the research ongoing in our group. |
Year(s) Of Engagement Activity | 2010 |
Description | Invited talk, 2013 Gordon Research Conference on Chromosome Dynamics, Il Ciocco, Lucca, Italy, 2013 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | I gave a talk on our work on chromosome segregation in archaea. The talk sparked questions and discussion. Becoming more visible as a scientist to an international audience. |
Year(s) Of Engagement Activity | 2013 |
Description | Invited talk, 2015 Gordon Research Conference on Chromosome Dynamics, Waterville Valley, New Hampshire |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Study participants or study members |
Results and Impact | This talk was given to experts in the field of genome biology and presented new data that were very well received. |
Year(s) Of Engagement Activity | 2015 |
URL | http://www.grc.org/programs.aspx?id=12469 |
Description | Invited talk, Department of Biology and Biotechnologies, University of Pavia, Italy, 2013. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | I gave a talk on our published work on chromosome segregation in archaea (BBRSC). Promotion of my research |
Year(s) Of Engagement Activity | 2013 |
Description | Invited talk, University of Birmingham |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Study participants or study members |
Results and Impact | Colleagues from the Institute of Microbiology and Infection attended the talk. |
Year(s) Of Engagement Activity | 2014 |
Description | Invited talk, University of Toulouse, Toulouse, France, 2013. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | The talk sparked questions and discussion. Promotion of my research to an international audience |
Year(s) Of Engagement Activity | 2013 |
Description | Molecular Biology of Archaea Conference (Cambridge) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | We presented a poster on DNA segregation in archaea. It sparked discussions and questions. Promotion of the research on archaea funded by the BBSRC grant. |
Year(s) Of Engagement Activity | 2010 |
Description | Molecular Biology of Archaea Workshop (Nottingham) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | I presented my work on the archaea project funded by the BBSRC. I guess the talk promoted my research to an audience of colleagues, postdocs and PhD students from other universities in the UK. |
Year(s) Of Engagement Activity | 2009 |
Description | Plasmid Biology 2008 Conference (Gdansk, Poland) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | Poster presentation Disseminating the data generated through my research |
Year(s) Of Engagement Activity | 2008 |
Description | Talk, 13th UK Meeting on Archaea, Nottingham, January 8-9, 2015 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Study participants or study members |
Results and Impact | The UK archaeal community attended this meeting. |
Year(s) Of Engagement Activity | 2015 |
URL | http://www.nottingham.ac.uk/conference/fac-mhs/lifesciences/archaea/programme/programme.aspx |
Description | UCAS days for undergraduate recruitment |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Talk to perspective undergraduate students Trying to enthuse the next generation of microbiologits. |
Year(s) Of Engagement Activity | 2007,2008,2009,2010,2011,2012 |