--- OA_place: publisher _id: '20741' abstract: - lang: eng text: "Life on Earth emerged when biomacromolecules were membrane-enclosed in a confined space where many essential chemical reactions were more likely to happen and thereby accelerate evolution. These kinds of membranes separated internal reactions from the outside chaos while staying flexible so that those primordial cells can move, adopt their shape and, most importantly, propagate. Such membrane plasticity still remains a defining feature of all modern cell types. This remarkable ability to change their shape is most prominently observed during their propagation (i.e., cell division). Throughout division, a cell undergoes drastic change in its shape, usually at the middle of the cell, pulling the two opposite membrane sides inward, closer to each other, and, finally, culminating in pinching off to separate the cell into two daughter cells. To achieve this, a cell needs to employ a protein machinery, usually termed divisome, that can coordinate all necessary intracellular processes with membrane remodelling and synthesis of other extracellular structures that decorate a cell. The focus of this dissertation is a membrane-remodelling FtsZ system that is present across all domains of life. FtsZ forms filaments that further self-organize into ring-like structures at the cell septum and together with other division proteins perform cell envelope synthesis and constriction. However, there are still knowledge gaps in our mechanistic understanding of division in both archaea and bacteria. My work presented in this dissertation centres around a simple yet not well understood question: How is the divisome positioned correctly at the mid-cell? To achieve the proper positioning, the divisome needs to (i) be recruited to the mid-cell and (ii) localized orthogonally to the long cell axis. I tackle these processes in two different systems by applying an in vitro biochemical bottom-up reconstitution approach. I use purified components of Haloferax volcanii and Escherichia coli divisome to explore how divisome is recruited to the mid-cell in archaea and how the Z-ring positions orthogonally to the long cell axis in bacteria, respectively. \r\n\r\nFirstly, I collaborate with archaeal cell and structural biologists to explore the assembly of early division proteins in two FtsZ-containing archaeon H. volcanii, a standard model system for understudied archaeal organisms. I particularly address the hierarchy of interactions that allow a tripartite complex formation (SepF-CdpB1-CdpB2) and how the hierarchy of interactions ultimately leads to the recruitment of FtsZ filaments to the septum. This part of work has been published in (Nußbaum et al., 2024). In collaboration with evolutionary biologists, I shed light on ancient features that archaeal divisome has retained to this day and also speculate on a property that it might have lost during the course of evolution. \r\n\r\nNext, I switch my attention to E. coli divisome. Particularly, I address the FtsZ’s intrinsic biophysical property that drives the Z-ring diameter, and thereby the perpendicular orientation of the Z-ring to the long cell axis based on suggested membrane curvature sensing mechanism (Vanhille-Campos et al., 2024). This property allows formation of different Z-ring diameters that match the variety of cell diameters present in prokaryotes. The results showcase that the distribution of charged amino acids in the intrinsically disordered linker at the C-terminus (CTL) of FtsZ is the major determining factor of Z-ring diameter with inter-CTL interactions as an underlying mechanism. \r\n\r\nFinally, I thoroughly explain the methodology I used to address the abovementioned projects, and I finish with a discussion on how early archaeal divisome assembly and curvature sensing mechanism in bacteria, at first sight unrelated topics, are interconnected and important groundwork for both fundamental and translational research. " acknowledged_ssus: - _id: Bio alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Marko full_name: Kojic, Marko id: 73e7ecd4-dc85-11ea-9058-88a16394b160 last_name: Kojic orcid: 0000-0001-7244-8128 citation: ama: Kojic M. Towards understanding the assembly mechanisms of the Z-ring in Archaea and Bacteria. 2025. doi:10.15479/AT-ISTA-20741 apa: Kojic, M. (2025). Towards understanding the assembly mechanisms of the Z-ring in Archaea and Bacteria. Institute of Science and Technology Austria. https://doi.org/10.15479/AT-ISTA-20741 chicago: Kojic, Marko. “Towards Understanding the Assembly Mechanisms of the Z-Ring in Archaea and Bacteria.” Institute of Science and Technology Austria, 2025. https://doi.org/10.15479/AT-ISTA-20741. ieee: M. Kojic, “Towards understanding the assembly mechanisms of the Z-ring in Archaea and Bacteria,” Institute of Science and Technology Austria, 2025. ista: Kojic M. 2025. Towards understanding the assembly mechanisms of the Z-ring in Archaea and Bacteria. Institute of Science and Technology Austria. mla: Kojic, Marko. Towards Understanding the Assembly Mechanisms of the Z-Ring in Archaea and Bacteria. Institute of Science and Technology Austria, 2025, doi:10.15479/AT-ISTA-20741. short: M. Kojic, Towards Understanding the Assembly Mechanisms of the Z-Ring in Archaea and Bacteria, Institute of Science and Technology Austria, 2025. corr_author: '1' date_created: 2025-12-09T13:08:11Z date_published: 2025-12-09T00:00:00Z date_updated: 2026-04-07T12:27:58Z day: '09' ddc: - '572' degree_awarded: PhD department: - _id: GradSch - _id: MaLo doi: 10.15479/AT-ISTA-20741 file: - access_level: closed checksum: a3643d07e93134b2490a566b02a4517d content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: mkojic date_created: 2025-12-10T13:09:58Z date_updated: 2025-12-10T13:09:58Z file_id: '20774' file_name: 2025_marko_kojic_thesis.docx file_size: 142876975 relation: source_file - access_level: open_access checksum: 0a096f0af6ccc3a8329d5bb8797ad533 content_type: application/pdf creator: mkojic date_created: 2025-12-10T13:09:38Z date_updated: 2025-12-10T13:09:38Z file_id: '20775' file_name: 2025_marko_kojic_thesis.pdf file_size: 8597045 relation: main_file success: 1 file_date_updated: 2025-12-10T13:09:58Z has_accepted_license: '1' language: - iso: eng license: https://creativecommons.org/licenses/by/4.0/ month: '12' oa: 1 oa_version: Published Version publication_identifier: isbn: - 978-3-99078-073-2 issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria related_material: record: - id: '15118' relation: part_of_dissertation status: public status: public supervisor: - first_name: Martin full_name: Loose, Martin id: 462D4284-F248-11E8-B48F-1D18A9856A87 last_name: Loose orcid: 0000-0001-7309-9724 title: Towards understanding the assembly mechanisms of the Z-ring in Archaea and Bacteria tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: dissertation user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd year: '2025' ... --- _id: '15118' abstract: - lang: eng text: Cell division in all domains of life requires the orchestration of many proteins, but in Archaea most of the machinery remains poorly characterized. Here we investigate the FtsZ-based cell division mechanism in Haloferax volcanii and find proteins containing photosynthetic reaction centre (PRC) barrel domains that play an essential role in archaeal cell division. We rename these proteins cell division protein B 1 (CdpB1) and CdpB2. Depletions and deletions in their respective genes cause severe cell division defects, generating drastically enlarged cells. Fluorescence microscopy of tagged FtsZ1, FtsZ2 and SepF in CdpB1 and CdpB2 mutant strains revealed an unusually disordered divisome that is not organized into a distinct ring-like structure. Biochemical analysis shows that SepF forms a tripartite complex with CdpB1/2 and crystal structures suggest that these two proteins might form filaments, possibly aligning SepF and the FtsZ2 ring during cell division. Overall our results indicate that PRC-domain proteins play essential roles in FtsZ-based cell division in Archaea. acknowledged_ssus: - _id: LifeSc acknowledgement: We thank X. Ye (ISTA) for providing the His–SUMO expression plasmid pSVA13429. pCDB302 was a gift from C. Bahl (Addgene plasmid number 113673; http://n2t.net/addgene:113673; RRID Addgene_113673). We thank B. Ahsan, G. Sharov, G. Cannone and S. Chen from the Medical Research Council (MRC) LMB Electron Microscopy Facility for help and support. We thank Scientific Computing at the MRC LMB for their support. We thank L. Trübestein and N. Krasnici of the protein service unit of the ISTA Lab Support Facility for help with the SEC coupled with multi-angle light scattering experiments. We thank D. Grohmann and R. Reichelt from the Archaea Centre at the University of Regensburg for providing the P. furiosus cell material. P.N. and S.-V.A. were supported by a Momentum grant from the Volkswagen (VW) Foundation (grant number 94933). D.K.-C. and D.B. were supported by the VW Stiftung ‘Life?’ programme (to J.L.; grant number Az 96727) and by the MRC, as part of UK Research and Innovation (UKRI), MRC file reference number U105184326 (to J.L.). N.T. and S.G. acknowledge support from the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant number ANR-10-LABX-62-IBEID), and the computational and storage services (Maestro cluster) provided by the IT department at Institut Pasteur. M.K. and M.L. were supported by the Austrian Science Fund (FWF) Stand-Alone P34607. For the purpose of open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any author accepted manuscript version arising. article_processing_charge: No article_type: original author: - first_name: Phillip full_name: Nußbaum, Phillip last_name: Nußbaum - first_name: Danguole full_name: Kureisaite-Ciziene, Danguole last_name: Kureisaite-Ciziene - first_name: Dom full_name: Bellini, Dom last_name: Bellini - first_name: Chris full_name: Van Der Does, Chris last_name: Van Der Does - first_name: Marko full_name: Kojic, Marko id: 73e7ecd4-dc85-11ea-9058-88a16394b160 last_name: Kojic orcid: 0000-0001-7244-8128 - first_name: Najwa full_name: Taib, Najwa last_name: Taib - first_name: Anna full_name: Yeates, Anna last_name: Yeates - first_name: Maxime full_name: Tourte, Maxime last_name: Tourte - first_name: Simonetta full_name: Gribaldo, Simonetta last_name: Gribaldo - first_name: Martin full_name: Loose, Martin id: 462D4284-F248-11E8-B48F-1D18A9856A87 last_name: Loose orcid: 0000-0001-7309-9724 - first_name: Jan full_name: Löwe, Jan last_name: Löwe - first_name: Sonja Verena full_name: Albers, Sonja Verena last_name: Albers citation: ama: Nußbaum P, Kureisaite-Ciziene D, Bellini D, et al. Proteins containing photosynthetic reaction centre domains modulate FtsZ-based archaeal cell division. Nature Microbiology. 2024;9(3):698-711. doi:10.1038/s41564-024-01600-5 apa: Nußbaum, P., Kureisaite-Ciziene, D., Bellini, D., Van Der Does, C., Kojic, M., Taib, N., … Albers, S. V. (2024). Proteins containing photosynthetic reaction centre domains modulate FtsZ-based archaeal cell division. Nature Microbiology. Springer Nature. https://doi.org/10.1038/s41564-024-01600-5 chicago: Nußbaum, Phillip, Danguole Kureisaite-Ciziene, Dom Bellini, Chris Van Der Does, Marko Kojic, Najwa Taib, Anna Yeates, et al. “Proteins Containing Photosynthetic Reaction Centre Domains Modulate FtsZ-Based Archaeal Cell Division.” Nature Microbiology. Springer Nature, 2024. https://doi.org/10.1038/s41564-024-01600-5. ieee: P. Nußbaum et al., “Proteins containing photosynthetic reaction centre domains modulate FtsZ-based archaeal cell division,” Nature Microbiology, vol. 9, no. 3. Springer Nature, pp. 698–711, 2024. ista: Nußbaum P, Kureisaite-Ciziene D, Bellini D, Van Der Does C, Kojic M, Taib N, Yeates A, Tourte M, Gribaldo S, Loose M, Löwe J, Albers SV. 2024. Proteins containing photosynthetic reaction centre domains modulate FtsZ-based archaeal cell division. Nature Microbiology. 9(3), 698–711. mla: Nußbaum, Phillip, et al. “Proteins Containing Photosynthetic Reaction Centre Domains Modulate FtsZ-Based Archaeal Cell Division.” Nature Microbiology, vol. 9, no. 3, Springer Nature, 2024, pp. 698–711, doi:10.1038/s41564-024-01600-5. short: P. Nußbaum, D. Kureisaite-Ciziene, D. Bellini, C. Van Der Does, M. Kojic, N. Taib, A. Yeates, M. Tourte, S. Gribaldo, M. Loose, J. Löwe, S.V. Albers, Nature Microbiology 9 (2024) 698–711. date_created: 2024-03-17T23:00:58Z date_published: 2024-03-04T00:00:00Z date_updated: 2026-04-07T12:27:57Z day: '04' department: - _id: MaLo doi: 10.1038/s41564-024-01600-5 external_id: isi: - '001183270800021' pmid: - '38443575' intvolume: ' 9' isi: 1 issue: '3' language: - iso: eng month: '03' oa_version: None page: 698-711 pmid: 1 project: - _id: fc38323b-9c52-11eb-aca3-ff8afb4a011d grant_number: P34607 name: In vitro reconstitution of bacterial cell division publication: Nature Microbiology publication_identifier: eissn: - 2058-5276 publication_status: published publisher: Springer Nature quality_controlled: '1' related_material: record: - id: '20741' relation: dissertation_contains status: public scopus_import: '1' status: public title: Proteins containing photosynthetic reaction centre domains modulate FtsZ-based archaeal cell division type: journal_article user_id: 317138e5-6ab7-11ef-aa6d-ffef3953e345 volume: 9 year: '2024' ...