Biology subjects

Bouet, J.-Y.

Publications and source records attributed to Bouet, J.-Y..

12 recordsLinked to original sources

bioRxiv2025

Excitable dynamics of ParA during bacterial cell division

ParABS system is a conserved machinery that drives the partitions of genome and other large intracellular cargos in bacteria. It works as a ParA concentration-based Brownian ratchet, which ensures the partition fidelity by operating at the critical point in its parameter space and localizing the ParA nearby to buffer against the large fluctuations in the ParA intracellular concentration. Despite these progresses, our understanding of the ParABS-driven partition mechanism remains incomplete. It is not understood what causes the large cell-to-cell fluctuation in the ParA intracellular concentration and why the bacterium cannot minimize this noise to better control the partition fidelity. We present experimental evidence with low-copy-number F-plasmids in E. coli that ParA robustly oscillates from pole to pole along the nucleoid length, underlying the observed large fluctuations in ParA intracellular concentration. Our theory-experiment synergy demonstrates that the ParA oscillation hinges on the nucleoid dimension and the ParB-mediated negative feedback with ParA in nucleoid binding. We suggest that the low-copy-number plasmid manages its partition fidelity with "autonomy": The plasmid harnesses the same ParB-mediated negative feedback - that dissociates the ParA from the nucleoid underneath the plasmid - to maintain the nucleoid-bound ParA at a proper level further away. And the ParA oscillation is reminiscent of this underlying feedback mechanism, which adapts the partition fidelity to the nucleoid growth. Our work may provide an overarching framework of intracellular partitioning in bacteria. Significance StatementParABS system is a conserved partition machinery for genome and other large cargos inside bacteria and works as a ParA concentration-based Brownian ratchet. It operates at the critical point in parameter space for sensitive adaptation and localizes the ParA nearby to buffer against the large cell-to-cell ParA concentration variations evidenced in bacteria. Why cannot the bacterium minimize this noise to better control the partition fidelity? Hereby, we showed that the large cell-to-cell ParA concentration variations stem from the ParA pole-to-pole oscillations along the nucleoid length and embodies an automony strategy that the intracellular cargo controls the nucleoid-bound ParA at a proper level to ensures its partition fidelity. Our work may provide an overarching framework of intracellular partitioning in bacteria.

biophysics↗

bioRxiv2025

Fast assembly and in vivo coalescence of ParB biocondensates involved in bacterial DNA partition

Faithful DNA segregation in bacteria relies on ParABS systems, in which ParB assembles into condensates at centromere-like parS sites, while the ATPase ParA spatially organizes these complexes. How these ParB condensates maintain dynamic behavior without collapsing into a single structure has remained unclear. Here, we combine chromosome degradation with quantitative imaging to dissect the kinetics and physical principles governing ParB condensate dynamics in vivo. In the absence of the nucleoid, ParB condensates from the plasmid F diffuse freely and coalesce within seconds upon encounter. Strikingly, quantitative analyses indicate that condensates may operate near the fusion-separation boundary, such that minimal energy is sufficient to split them after replication, preventing irreversible coalescence. Using mutants, we demonstrate that condensate assembly is required for coalescence and uncover a dual role for ParAF: nucleoid tethering restricts condensate mobility and limits fusion, while ParAF also promotes a ParBF state competent for assembly and coalescence, likely by enhancing ParB-ParB interactions. Finally, condensates rapidly disassemble and reassemble upon 1,6-hexanediol treatment, underscoring their reversibility and the stabilizing contribution of ParB-DNA interactions. Together, our results establish ParBF complexes as bona-fide biocondensates tuned by ParAF to ensure robust DNA segregation. More broadly, these findings highlight regulated phase separation as a key organizing principle of bacterial replicons.

microbiology↗

bioRxiv2025

PacL-organized membrane-associated effluxosomes coordinate multi-metal resistance in Mycobacterium tuberculosis

Metal ion homeostasis is crucial for bacterial pathogens to withstand metal-induced stress during infection. However, the mechanisms underlying bacterial resistance to metal stress remain incompletely understood, particularly how bacteria coordinate responses to simultaneous exposure to multiple metals. Here, we uncover a previously unrecognized mechanism by which Mycobacterium tuberculosis, the causative agent of tuberculosis, orchestrates a coordinated response to multi-metal stress. We demonstrate that M. tuberculosis assembles dynamic, membrane-associated platforms, organized by PacL proteins, that confer resistance to multiple metals simultaneously. PacL proteins function as scaffolds, clustering multiple P-type ATPase (P-ATPase) pumps, CtpC, CtpG, and CtpV, into functional complexes we term "effluxosomes". Our findings show that PacL proteins are critical for stabilizing CtpG within membrane-associated clusters, conferring cadmium tolerance, while CtpC serves as a backup, promoting cross-resistance to both zinc and cadmium. Using super-resolution microscopy and single-particle tracking, we elucidate the 3D structure and dynamics of effluxosomes in the mycobacterial membrane. We further demonstrate that conserved residues within the transmembrane domain of PacL proteins are crucial for the assembly of dynamic effluxosomes, which are essential for P-ATPase activity. Additionally, we reveal that PacL1 exhibits metallochaperone activity, binding zinc, cadmium, and copper via a conserved C-terminal motif. Proximity labeling further identifies an extensive PacL1 interaction network, encompassing multiple proteins involved in stress adaptation. Our findings introduce effluxosomes as dynamic, membrane-associated efflux machineries that mediate coordinated multi-metal resistance in M. tuberculosis, providing new insights into bacterial metal homeostasis and unveiling potential antimicrobial targets.

microbiology↗

bioRxiv2025

The ParB-CTP Cycle Activates Phase Separation in Bacterial DNA Segregation

Cell function relies on liquid-like membraneless organelles formed through phase transitions, yet the mechanisms ensuring their specificity and rapid assembly remain poorly understood. In bacterial chromosome segregation via the ParABS system, hundreds of ParB proteins are recruited around the centromere-like parS sequence forming the partition complex. Recent studies have shown that ParB binds CTP and undergoes cycles of loading and unloading near parS, however, this accounts for the recruitment of only a small fraction of ParB molecules, leaving its role unclear. Separately, a lattice gas model with fixed interaction energy has been proposed to describe ParB phase separation, but it fails to explain key experimental observations, including the absence of droplets in ParB variants. We reconcile these two perspectives by proposing that the ParB-CTP cycle acts as a molecular switch that enhances ParB-ParB interactions, triggering phase transition from vapor to liquid-like condensates. Our hypothesis is supported by numerical simulations of droplet formation and experiments showing that ParB variants disrupting the CTP cycle fail to undergo phase separation. These findings establish a mechanistic framework for ParB-CTP-mediated phase transitions and may have broader implications for understanding the spatial control of intracellular condensate formation.

cell biology↗

bioRxiv2024

In vivo Assembly of Bacterial Partition Condensates on Supercoiled and Linear DNA

In bacteria, faithful DNA segregation of chromosomes and plasmids is mainly mediated by ParABS systems. These systems, consisting of a ParA ATPase, a DNA binding ParB CTPase, and centromere sites parS, orchestrate the separation of newly replicated DNA copies and their intracellular positioning. Accurate segregation relies on the assembly of a high-molecular-weight complex, comprising a few hundreds of ParB dimers nucleated from parS sites. This complex assembles in a multi-step process and exhibits dynamic liquid-droplet properties. Despite various proposed models, the complete mechanism for partition complex assembly remains elusive. This study investigates the impact of DNA supercoiling on ParB DNA binding profiles in vivo, using the ParABS system of the plasmid F. We found that variations in DNA supercoiling does not significantly affect any steps in the assembly of the partition complex. Furthermore, physical modeling, leveraging ChIP-seq data from linear plasmids F, suggests that ParB sliding is restricted to approximately 2-Kbp from parS, highlighting the necessity for additional mechanisms beyond ParB sliding over DNA for concentrating ParB into condensates nucleated at parS. Lastly, explicit simulations of a polymer coated with bound ParB suggest a dominant role for ParB-ParB interactions in DNA compaction within ParB condensates.

molecular biology↗

bioRxiv2021

Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

Transition metals are toxic at high concentrations. The P1B-ATPase metal exporter CtpC/Rv3270 is required for resistance to zinc poisoning in the human pathogen Mycobacterium tuberculosis. Here, we discovered that zinc resistance also depends on the chaperone-like protein PacL1/Rv3269. PacL1 bound Zn2+, but unlike PacL1 and CtpC, the PacL1 metal-binding motif (MBM) was required only at high zinc concentrations. PacL1 co-localized with CtpC in dynamic microdomains within the mycobacterial plasma membrane. Microdomain formation did not require flotillins nor the PacL1 MBM. Instead, loss of the PacL1 Glutamine/Alanine repeats led to loss of CtpC and sensitivity to zinc. PacL1 and CtpC are within the same operon, and homologous PacL1-P1B-ATPase pairs are widely distributed within and across prokaryotes. PacL1 colocalized and functioned redundantly with PacL orthologs in Mycobacterium tuberculosis. Overall, our study suggests that PacL proteins are scaffolds that assemble P-ATPase-containing metal efflux platforms, a novel type of functional membrane microdomain that underlies bacterial resistance to metal poisoning.

microbiology↗

bioRxiv2021

Three ParA dimers cooperatively assemble on type Ia partition promoters

Accurate DNA segregation is essential for faithful inheritance of genetic material. In bacteria, this process is mainly ensured by a partition system (Par) composed of two proteins, ParA and ParB, and a centromere site. The auto-regulation of Par operon expression is important for efficient partitioning, and is primarily mediated by ParA for type Ia plasmid partition systems. For the plasmid F, four ParAF monomers were proposed to bind to four repeated sequences in the promoter region. By contrast, using quantitative surface plasmon resonance, we showed that three ParAF dimers bind to this region. We uncovered that one perfect inverted repeat (IR) motif, consisting of two hexamer sequences spaced by 28-bp, constitutes the primary ParAF DNA binding site. A similar but degenerated motif overlaps the former. ParAF binding to these motifs is well supported by biochemical and modeling analyses. In addition, molecular dynamics simulations predict that the winged-HTH domain displays high flexibility, which may favor the cooperative ParA binding to the promoter region. We propose that three ParAF dimers bind cooperatively to overlapping motifs thus covering the promoter region. A similar organization is found on both closely related and distant plasmid partition systems, suggesting that such promoter organization for auto-regulated Par operons is widespread and may have evolved from a common ancestor.

biochemistry↗

bioRxiv2020

Physical modeling of a sliding clamp mechanism for the spreading of ParB at short genomic distance from bacterial centromere sites

Bacterial ParB partitioning proteins involved in chromosomes and low-copy-number plasmid segregation have recently been shown to belong to a new class of CTP-dependent molecular switches. Strikingly, CTP binding and hydrolysis was shown to induce a conformational change enabling ParB dimers to switch between an open and a closed conformation. This latter conformation clamps ParB dimers on DNA molecules, allowing their diffusion in one dimension along the DNA. It has been proposed that this novel sliding property may explain the spreading capability of ParB over more than 10-Kb from parS centromere sites where ParB is specifically loaded. Here, we modeled such a mechanism as a typical reaction-diffusion system and compared this Clamping & sliding model to the ParB DNA binding pattern from high-resolution ChIP-sequencing data. We found that this mechanism cannot account for all the in vivo characteristics, especially the long range of ParB binding to DNA. In particular, it predicts a strong effect from the presence of a roadblock on the ParB binding pattern that is not observed in ChIP-seq. Moreover, the rapid assembly kinetics observed in vivo after the duplication of parS sites is not easily explained by this mechanism. We propose that Clamping & sliding might explain the ParB spreading pattern at short distances from parS but that another mechanism must apply for ParB recruitment at larger genomic distances.

systems biology↗

bioRxiv2020

Spatial control over near-critical-point operation ensures fidelity of ParABS-mediated bacterial genome segregation

In bacteria, most low-copy-number plasmid and chromosomally encoded partition systems belong to the tripartite ParABS partition machinery. Despite the importance in genetic inheritance, the mechanisms of ParABS-mediated genome partition are not well understood. Combining theory and experiment, we provided evidences that the ParABS system - partitioning via the ParA gradient-based Brownian ratcheting - operates near a critical point in vivo. This near-critical-point operation adapts the segregation distance of replicated plasmids to the half-length of the elongating nucleoid, ensuring both cell halves to inherit one copy of the plasmids. Further, we demonstrated that the plasmid localizes the cytoplasmic ParA to buffer the partition fidelity against the large cell-to-cell fluctuations in ParA level. Thus, the spatial control over the near-critical-point operation not only ensures both sensitive adaption and robust execution of partitioning, but sheds light on the fundamental question in cell biology: How do cells faithfully measure cellular-scale distance by only using molecular-scale interactions?

microbiology↗

bioRxiv2019

Role of centromere sites in activation of ParB proteins for partition complex assembly

The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. We conclude that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.

microbiology↗

bioRxiv2019

ATP-driven separation of liquid phase condensates in bacteria

Liquid-liquid phase separated (LLPS) states are key to compartmentalise components in the absence of membranes, however it is unclear whether LLPS condensates are actively and specifically organized in the sub-cellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB) and a motor (ParA). We show that parS-ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume, but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favoured by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates and localises non-canonical LLPS condensates in the sub-cellular space.

cell biology↗

bioRxiv2018

Robust and conserved stochastic self-assembly mechanism for dynamic ParB-parS partition complexes on bacterial chromosomes and plasmids

Chromosome and plasmid segregation in bacteria are mostly driven by ParABS systems. These DNA partitioning machineries rely on large nucleoprotein complexes assembled on centromere sites (parS). However, the mechanism of how a few parS-bound ParB proteins nucleate the formation of highly concentrated ParB clusters remains unclear despite several proposed physico-mathematical models. We discriminated between these different models by varying some key parameters in vivo using the plasmid F partition system. We found that Nucleation & caging is the only coherent model recapitulating in vivo data. We also showed that the stochastic self-assembly of partition complexes (i) does not directly involve ParA, (ii) results in a dynamic structure of discrete size independent of ParB concentration, and (iii) is not perturbed by active transcription but is by protein complexes. We refined the Nucleation & Caging model and successfully applied it to the chromosomally-encoded Par system of Vibrio cholerae, indicating that this stochastic self-assembly mechanism is widely conserved from plasmids to chromosomes.

systems biology↗