Thursday, 15th December, 2022
Hicham Sekkouri Alaoui will present their research on study of study of the self-assembly mechanism of the main bacterial DNA partition system.
Abstract :
My project concerns the ParBS nucleoprotein self-assembly involved in chromosomes and low copy number plasmids partitioning in most bacteria. Recent discoveries revealed that this system is catalyzed by CTP binding to the centromere-binding-protein ParB (Osorio-Valeriano et al., 2019; Soh et al., 2019). Indeed, this event is essential for accurate intracellular positioning of thousands of ParB around a few parS centromere sites. However, the self-assembly mechanism of this droplet-like structures remains speculative (Guilhas et al., 2020a), and a recent physico-mathematical modèle predicts an effect of DNA supercoiling on the ParB DNA binding pattern (Walter et al., 2021). Based on ChIP-sequencing experiments, the main aims of my Ph.D. project are (i) to test in vivo this effect on the ParB self-assembly model and (ii) to understand ParB propagation behavior from parS.
For this purpose, I gathered a set of strains probed to reflect a wide range of DNA supercoiling density (σ). Indeed, I found that E. coli topA, E. coli wt, S. thy LT2 wt and LT2 gyrB offer a free DNA σ variation that goes from -0,038 to -0,026%. In this range, the result of ParB binding pattern on the F plasmid is mainly conserved which confirms the prediction relevance. To engender the zero-supercoiling condition, I generated bacterial strains carrying F plasmids linearized by telomerization (Deneke et al., 2000) in ParB propagation region. By contrast to the expected immediate signal drop, the ChIP-sequencing results indicate that DNA supercoiling density variation does not affect the ParB DNA binding pattern up to the telomeres position. Such robustness supports the idea of a synergic network of ParB interactions centered at parS (Debaugny et al., 2018; Sanchez et al., 2015).
Thesis defense will be preceded by a seminar: Dr Olivier Espeli (Collège de France, Paris) : “Strategies allowing E. coli associated with Crohn’s disease to survive within immune cells”
GeDy team
Friday, 4th November, 2022
Alix Meunier will present their research on study of coordination between growth and cell cycle in Escherichia coli.
Abstract :
Like all living things, bacteria need to reproduce to survive in the long term. Most bacteria do so by binary fission, including the model organism Escherichia coli. Underneath its conceptual simplicity, binary fission appears to be a complex molecular process. It involves multiple basic mechanisms, which are highly conserved among strains of the same species. The efficiency of this proliferation program, i.e. its rapidity and low failure rate, also relies on a strong spatio-temporal coordination of all the cell cycle events. This is particularly remarkable in the case of fast-growing bacteria that are able to manage several replication eyes simultaneously. Since the 1960’s, bacterial physiology studies have been working to decipher this molecular network and to define general models describing cell cycle coordination. Recent advances in single-cell analysis techniques offer new perspectives. I am particularly interested in the phenotypic diversity found between strains belonging to the same species. During my thesis, I applied a comparative cell biology approach to test the existing correlations between phenotypic parameters related to the cell cycle. Using optical microscopy, cell density monitoring, droplet digital PCR and flow cytometry, I accurately characterized a series of phenotypes, namely morphology, proportions of segregating and dividing cells during exponential growth, population growth rate, duration of cell cycle periods, for eight different E. coli strains. My data suggest differences in the correlation between cell size and cell growth, implying differences in the volume at the initiation of DNA replication between strains of the same bacterial species. They also reveal a remarkable conservation of genetic replication and cell division durations between these strains. The segregation period of chromosomal replicates also appears to be relatively invariant for a given strain under different growth conditions, but its duration varies from strain to strain. Thus, a diversity in the control of the initiation time, but also in the duration of some cell cycle events can be identified between strains sharing more than 95% identity between the genes of their core genome. These observations could lead to the identification of new factors involved in the control of the bacterial cell cycle.
Thesis defense will be preceded by a seminar: Pr Erick Denamur (Paris Cité and Sorbonne Paris Nord Universities) : “Population genetics of Escherichia coli natural isolates.”
GeDy team
Friday, 23th September, 2022
Luis Orenday Tapia will present their research on study of Mechanisms of biogenesis and maintenance of the outer membrane of Gram-negative bacteria.
Abstract :
Gram-negative bacteria encompass many multidrug resistant pathogens. Their multilayered envelope is formed by an inner membrane (IM), an outer membrane (OM), and a separating periplasm containing the peptidoglycan (PG). The OM forms a semipermeable barrier that prevents the entry of numerous chemicals. Lipoproteins and integral OM proteins (OMPs) are crucial components of the OM that exchange nutrients, excrete toxic molecules and interact with the environment. The OM contains lipopolysaccharide (LPS) in the external leaflet and phospholipids in the internal one. All OM components are synthesized in the cytosol or at the IM and are delivered to the OM by specific transport pathways. Among these, the β-barrel assembly machinery (BAM) complex folds and inserts OMPs into the OM. The assembly of some OMPs requires the poorly understood activity of the translocation and assembly module (TAM). The accumulation of unfolded OMPs in the periplasm triggers activation of the σE-mediated envelope stress response. σE regulates a number of genes enhancing the levels of BAM subunits. Whereas many envelope biogenesis pathways have been described in the last decades, little is known about how these processes are coordinated during the life cycle of the cell.
This PhD project aimed at studying the regulation of BAM by determining all BAM interactions in the envelope of the enterobacterium Escherichia coli. We setup a quantitative proteomic approach to analyze the BAM complex purified upon mild-solubilization of the cell envelope. The identified BAM putative interactors include two key envelope proteins, the division and OM-stress associated lipoprotein DolP/YraP and the IM-anchored transporter protein TamB. During my PhD defense on the 23rd of september 2022, I will present my results on how the interplay of BAM with both DolP and TamB contributes to form and preserve an effective semipermeable barrier that protects bacteria from stress and noxious molecules including antibiotics.
Thesis defense will be preceded by 2 seminars of 30 minutes each:
– Peter van Ulsen (Vrije Universiteit, Amsterdam) : “Autotransporter Hbp as model, vehicle and target”
– Alessandra Polissi (Università di Milano) : “Peptidoglycan remodeling as a strategy to survive outer membrane stress in Escherichia coli “
Equipe Raffaele Ieva
Thursday, 24th March, 2022
Valentin Quèbre will present their research on study of the DNA/Proteins complexes involved in bacterial DNA segregation.
Abstract :
Bacterial chromosomes and low copy number plasmids segregation is based on an active positioning mechanism. It consists in the partition systems that ensures the proper intracellular positioning of replicons to be faithfully transmitted to the daughter cells. The partition systems involves three cis-encoded partners. A DNA binding protein (ParB), is assembled in partition complexes at centromeric sequences (parS). An NTPase, which interacts with the partition complex, drives the segregation process and allows the complexes, and thus the plasmids, to be properly positioned inside the cell. My Ph.D project focused first on the better understanding of the partition complex assembly of the widespread type I system of the F plasmid and pESBL. Then, to decipher the global mechanism of the partition process of the recently discovered atypical system on R388, which does not involve any plasmid encoded NTPase to ensure its intracellular positioning.
Thus, my project is divided in three parts, aiming to (i) understand by an mutational approach, the initiation mechanism for the self-assembly of the majority of F plasmid ParB in a dynamic high molecular weight complex around parS, (ii) identify the pESBL partition system partners, in vitro characterize the ParB/parS interaction profile and in silico determine the group to which it belongs, (iii) identify the roles of the different domains of the R388 DNA binding protein StbA in its activities and characterize the StbA interaction modalities on its centromere by high throughput sequencing and biochemical approaches, to understand the partition complex architecture.
This study allows us to improve our knowledge on the Type I partition system and to shed light on the DNA/protein interaction specificities of an atypical system, carried by broad-host-range plasmids, opening the way to a better understanding of DNA segregation mechanism..
Gedy team- Jean-Yves Bouet and François Cornet
Mickaël Maziero will present their research on the competence induction pathways caracterisation for Streptococcus pneumoniae transformation.
Abstract :
Streptococcus pneumoniae is a pathogenic bacterium commensal of human nasopharynx. This bacterium is involved in multiple pathologies such as pneumonia, otitis media and meningitis. According to the world health organisation, it is responsible for several million deaths per year. This bacterium poses many problems due to its adaptability skill based on its ability to perform natural genetic transformation. Natural transformation allows cells to capture exogenous DNA in the external environment, internalize it into the cytoplasm and recombine it into the chromosome by homologous recombination. By this way, pneumococcus is able to easy acquire new genetic traits. This ability is transient and only takes place during a stress-induced physiological state called competence. The comCDE operon is the central element in triggering the competence state. The secreted peptide encoded by the comC gene is capable of activating the two-component regulatory system ComD/E. Sequentially at this activation, ComD/E which will auto-activate its own transcription, generating an positive autocatalytic loop and resulting in a rapid populational switch to competence. Due to its exponential propagating nature, competence must be tightly regulated. However, the external stimuli and molecular signalling pathways controlling competence development remain poorly characterized. The work of this thesis aims to address this issue. We have demonstrated that fever-level temperatures induce competence. We have characterized the molecular pathway linking this stress to the comCDE operon. We have also shown that this induction pathway is not the only one for competence stimulation and that at least one independent second exists and involves a toxin/antitoxin system cibling the cellular DNA replication machinery. The fact that there is an existence of multiple pathways converging on the comCDE operon strongly suggests that competence is a general stress regulon and that is highly linked to the pneumococcal cell cycle.
Patrice Polard team
Wednesday, 15th December 2021
Yiying Yang will present the results of her PhD work on the mechanisms of the biogenesis and maintenance of the outer membrane in Gram-negative bacteria.
Abstract:
Gram-negative bacteria include a number of dreadful animal pathogens that are particularly resistant to antibiotic therapies thanks to the sheltering function of their bacterial envelope. The envelope is composed of an inner and an outer membrane (IM and OM), and the separating periplasm containing the peptidoglycan (PG). The outer leaflet of the OM bilayer largely consists of lipopolysaccharide (LPS) that forms a permeability barrier against toxic molecules, including detergents and small hydrophobic molecules. Nutrients are transported via OM-spanning proteins (OMPs). Other OMPs perform envelope biogenesis functions, including the assembly of OMPs and LPS. OMPs are assembled into the OM by the beta-barrel assembly machinery (BAM), a heteropentamer containing the essential OMP BamA and four lipoproteins BamBCDE. The assembly of LPS requires another essential OMP, LptD, which stably associates with the lipoprotein LptE. Defective assembly of OMPs causes envelope stress and renders Gram-negative bacteria sensitive to antibiotics and detergents. Hence, the BAM complex represents a promising target for the development of new therapies.The mechanistic details of how the BAM complex functions ensuring efficient OM biogenesis are only marginally understood. By using a quantitative mass-spectrometry strategy the hosting lab has recently identified two novel putative interactors of the BAM complex of Escherichia coli, the lipoproteins DolP (formerly YraP) and BilB, both of unknown functions. The aim of this PhD thesiswork was to characterize the roles of DolP and BilB at the BAM complex.
Raffaele Ieva team
Monday, 13th December 2021
Elise Courtais will present the results of her PhD work on study of the cell division arrest induced during competence for natural genetic transformation in Streptococcus pneumoniae
Abstract :
Natural genetic transformation is a programmed mechanism of horizontal gene transfer in bacteria. It requires the entry of cells into a transient physiological state called competence, during which a large membrane-associated multiprotein complex is synthesized and ensures binding, internalization and integration by homologous recombination of exogenous DNA into the recipient genome. In the human pathogen Streptococcus pneumoniae, this machinery assembles at the cytoplasmic membrane and across the cell wall at midcell, the division site. In this species, competence develops in every cells of an exponentially growing culture and causes a growth arrest phenotype, suggesting a functional link between competence development and the cell cycle. During my thesis, I investigated the mechanism of this growth arrest.
I first tested the involvement of different components of the transformation machinery, localized at the division site, in the growth arrest observed during the induction of competence. This research allowed me to demonstrate that the assembly of the transformation pilus, responsible for the capture of exogenous DNA, leads to the aggregation of competent cells. My results suggest that this aggregation plays a role in fratricide, a process specific to S. pneumoniae in which competent cells develop the ability to “kill” non-competent sister cells.
I also adapted a super-resolution microscopy technique, with the aim of understanding the molecular mechanism of the inhibition of cell division in competent cells. I have developed tools allowing 3D- PALM (PhotoActivated Localization Microscopy) acquisitions, a microscopy technique based on single molecule localization with a resolution of about 50 nm, to determine the structure of the FtsZ ring in 3 dimensions. To study the fate of the FtsZ ring during the different stages of the cell cycle, I measured the morphological parameters of each cell from phase contrast images. My first results show that the FtsZ proteins form a heterogeneous ring with dimensions ranging from 300 to 700 nm in diameter, while the thickness (~110 nm) and depth (~100 nm) remain constant during the cell cycle. The presence of areas of high fluorescence intensity and areas of lower intensity indicate that the proteins are not homogeneously distributed in the ring but are grouped as clusters of varying density.
Patrice Polard team
Friday, 9th July 2021, 2 pm
Cyril Moulin, PhD student supported by the Ligue Contre le Cancer and the Fondation pour la Recherche Médicale, will present the results of his PhD work on the mechanisms of mitochondrial matrix and inner membrane protein biogenesis.
For this occasion, on the 6th of July starting from 2:30, Maria Bohnert (Münster University) and Thomas Becker (Bonn University) will present their research on the biogenesis and dynamics of cell organelles in two consecutive short presentations (CBI Seminar Bohnert and Becker)
Raffaele Ieva team
