Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / TIMMINS Joanna

Structure and dynamics of the nucleoid

In all organisms, genomic DNA is compacted several orders of magnitude in order to fit into a confined region of the cell (nuclei of eukaryotes or nucleoids of prokaryotes) and yet must remain accessible for essential DNA-related processes. In most cases, our understanding of bacterial cellular processes is more advanced than that of eukaryotes, which are typically more complex and challenging to decipher. However, this is not the case when it comes to the study of nucleoid organization and chromosome segregation, two essential processes, which are at the heart of many cellular processes. Numerous studies of eukaryotic chromatin have revealed its detailed molecular architecture and identified a wide range of factors involved in its regulation. In contrast, until recently, little was known regarding the organization of bacterial nucleoids, which were for many years believed to be mostly unstructured. This was due in part to the small size of bacterial nucleoids (<1µm3), making them difficult to study by classical optical microscopy.

In bacteria, packaging of genomic DNA is achieved by several mechanisms : DNA supercoiling, DNA compaction by nucleoid-associated proteins (NAPs), such as the histone-like protein HU, and several additional factors including molecular crowding and depletion forces. However, the detailed molecular mechanisms underlying DNA compaction, nucleoid organisation and chromosome segregation, three intimately linked processes, are still only poorly understood. We are using an integrated approach combining in vitro (Chen et. al., 2020) in silico (Hognon et. al., 2019) and in vivo (Floc’h et. al., 2019) studies to better apprehend these processes.

For example, in collaboration with the PIXEL team at IBS and the team of Fabrice Confalonieri at I2BC, Orsay, we recently demonstrated that the complex, multipartite genome of D. radiodurans forms a single nucleoid that is indeed highly condensed, but also, more surprisingly, very dynamic, adopting multiple distinct configurations as the bacterium progresses through its cell cycle. In addition to the previously reported toroidal shape, the nucleoid was notably seen to form elongated and branched structures at the late stages of the cell cycle that were found to specifically align perpendicular to the future division axis, a process reminiscent of eukaryotic chromosome alignment in metaphase. Such major conformational rearrangements of bacterial nucleoids had never been described before and clearly demonstrate that nucleoid organisation and cell cycle progression are tightly coupled.

Using a combination of genetics, biochemistry, atomic force microscopy, structural biology and advanced fluorescence microscopy, our goal is now to decipher the molecular mechanisms underlying this remarkable organisation and plasticity, and how they contribute to the outstanding radiation resistance phenotype of D. radiodurans.

Members of the team

• Anne-Sophie BANNEVILLE
• Salvatore DE BONIS
• Fabienne HANS
• Jean-Philippe KLEMAN
• Françoise LACROIX
• Joanna TIMMINS
• Pierre VAUCLARE

Techniques

• Molecular biology (cloning & genetic manipulation of D. radiodurans)
• Microbiology
• Physiology & Respiration
• UV and ionising radiation
• Recombinant expression and purification of proteins
• Biochemical & biophysical characterisation of protein-DNA complexes
• Macromolecular crystallography
• Confocal spinning-disk fluorescence microscopy
• Single-molecule localisation microscopy (PALM) and single-particle tracking PALM
• Fluorescence recovery after photobleaching (FRAP)
• Flow cytometry

Collaborations

• Dominique BOURGEOIS (PIXEL team, I2SR, IBS)
• Jean-Luc PELLEQUER (MEM Group, IBS)
• Irina GUTSCHE (MICA Group, IBS)
• Pascale SERVANT & Fabrice CONFALONIERI (I2BC, Orsay)
• François DEHEZ & Antonio MONARI (LPCT, Nancy)

Major publications

Chen SW, Banneville A-S, Teulon J-M, Timmins J and Pellequer J-L. Nanoscale surface structures of DNA bound to Deinococcus radiodurans HU unveiled by atomic force microscopy. Nanoscale (2020) 12, 22628. DOI : 10.1039/D0NR05320A.

Hognon C, Garaude S, Timmins J, Chipot C, Dehez F and Monari A. Molecular basis of DNA packaging in bacteria revealed by all-atoms molecular dynamic simulations : The case of histone-like proteins in Borrelia burgdorferi. J. Phys. Chem. Lett. (2019) 10, 7200-7207. DOI : 10.1021/acs.jpclett.9b02978.

Floc’h K, Lacroix F, Servant P, Wong YS, Kleman JP, Bourgeois D and Timmins J. Cell morphology and nucleoid dynamics in dividing D. radiodurans. Nat Commun. (2019) 10 (1). p.3815. DOI : 10.1038/s41467-019-11725-5.

Floc’h K, Lacroix F, Barbieri L, Servant P, Galland R, Butler C, Sibarita JB, Bourgeois D and Timmins J. Bacterial cell wall nanoimaging by autoblinking microscopy. Scientific Reports (2018) 8 (1) p. 14038. DOI : 10.1038/s41598-018-32335-z.