Imaging and modeling the Bacterial Mitosis – IBM

We are implementing or developing several microscopy methods: (i) fast, high-throughput epifluorescence microscopy, (ii) single-molecule super-resolution microscopy, and (iii) structured-illumination. These methodologies allow us to directly measure the spatial distribution of motor and centromere-binding proteins, their dynamics, and the motion of partition complexes relative to the motor.

We already publish three articles:
#1- Nous avons proposé un nouveau modèle d’assemblage du complexe de partition, publié dans la revue Cell Systems (http://www.cell.com/cell-systems/abstract/S2405-4712%2815%2900057-5).
La combinaison des approches de génétiques, biochimie et génome-wide avec la microscopie à super-résolution (PALM) couplée avec la modélisation physique
#2- La construction des plasmides et des souches ainsi que leur caractérisation fonctionnelle en microscopie en vue de leur utilisation future pour la validation du modèle d’assemblage du complexe de partition à donner lieu à publication (http://www.sciencedirect.com/science/article/pii/S0147619X15000323).
#3- L’observation pour la première fois en 3 dimensions du positionnement des acteurs de deux systèmes modèle de ségrégation au centre du nucléoïde bactérien, ainsi que la corrélation de ce positionnement avec les zones denses de l’ADN permet une avancée majeure dans la compréhension et la modélisation du processus de ségrégation de l’ADN bactérien. Ces travaux sont publiés dans la revue Nature Communications (http://www.nature.com/ncomms/2016/160705/ncomms12107/full/ncomms12107.html).

We have demonstrated for the first time that the partition components are all embedded within the bacterial nucleoid. This provides a completely different view of how the DNA is positionned and segregated in bacteria, and we are currently developping new physical model to fully explain this important step of the bacterial cell cycle.

- Sanchez et al., 2016 Cell Systems (http://www.cell.com/cell-systems/abstract/S2405-4712%2815%2900057-5).
- Diaz et al., 2016 Plasmids (http://www.sciencedirect.com/science/article/pii/S0147619X15000323).
- Le Gall et al., 2016 Nature Communications (http://www.nature.com/ncomms/2016/160705/ncomms12107/full/ncomms12107.html).

Bacterial proliferation relies on a series of essential processes occurring on the genome. Among them, DNA segregation remains enigmatic, mainly because bacterial cellular processes involved in chromosome dynamics (transcription, replication, recombination and segregation) overlap in time and take place in a single cellular compartment. Recent progress using genetics, fluorescence microscopy and biochemical methods led to the identification of the main actors involved in the process of DNA segregation in bacteria, but the molecular mechanisms are still largely unclear.
Bacterial plasmids and chromosomes encode active segregation systems to ensure stability (hereafter partition or Par). Par systems are minimalistic, encoding only three dedicated components that are necessary and sufficient for partition: a centromere site, a centromere-binding protein, and a motor ATPase. The latter protein self-organizes and patterns the bacterial nucleoid to spatially organize plasmids and chromosomes. The main limitations of previous studies were: (i) the inability to reproduce a functional in vitro reconstituted partition system, (ii) the small size of bacteria combined with limited sensitivity, and (iii) the spatio-temporal resolution of conventional fluorescence microscopy methods were inadequate to attain unambiguous observations of the segregation event.
The aim of this multidisciplinary project is to use and develop state-of-the-art advanced fluorescence microscopy and modeling methods to directly visualize and quantitatively characterize the process of bacterial DNA segregation at high spatial and temporal resolutions. These measurements, together with high-through put sequencing techniques, physical models and specific in vitro characterized mutants, will allow us for the first time to quantitatively dissect in vivo the process of DNA segregation.
Specifically, we will implement or develop several microscopy methods: (i) fast, high-throughput epifluorescence microscopy, (ii) single-molecule super-resolution microscopy, and (iii) structured-illumination. These methodologies will allow us to directly measure the spatial distribution of motor and centromere-binding proteins, their dynamics, and the motion of partition complexes relative to the motor. These measurements will allow us to discriminate between, or reconcile, the two main segregation models that are currently under intense debate: filament and diffusion ratchet based models.
We will work on the experimental model system that has been developed for more than fifteen years in Partner 1’s lab (LMGM, Toulouse): the E. coli plasmid F, which carries a classical Walker ATPase partition system. Whereas the principal approaches used by the group so far have been based on molecular genetics and biochemistry, we now intend to exploit recent developments in fluorescence microscopy and in single molecule visualization applied to bacteria to approach essential questions from different angles. To this goal, we have established collaborations with (i) Partner 2 at the CBS (Montpellier) who has developed super-resolution microscopy in bacteria, and is also investigating bacterial motors that segregate DNA, and (ii) Partner 3 at the LCC (Montpellier) who develops physical modeling of self-assembling and self-organizing phenomena in biological systems. Experiments will be performed at the LMGM and the CBS, and the modeling task at the LCC. The synergy between these three complementary groups bringing their own competence will be directed toward the understanding of the molecular mechanism responsible for DNA segregation in bacteria.