191 research outputs found
Physical descriptions of the bacterial nucleoid at large scales, and their biological implications.
Recent experimental and theoretical approaches have attempted to quantify the physical organization (compaction and geometry) of the bacterial chromosome with its complement of proteins (the nucleoid). The genomic DNA exists in a complex and dynamic protein-rich state, which is highly organized at various length scales. This has implications for modulating (when not directly enabling) the core biological processes of replication, transcription and segregation. We overview the progress in this area, driven in the last few years by new scientific ideas and new interdisciplinary experimental techniques, ranging from high space- and time-resolution microscopy to high-throughput genomics employing sequencing to map different aspects of the nucleoid-related interactome. The aim of this review is to present the wide spectrum of experimental and theoretical findings coherently, from a physics viewpoint. In particular, we highlight the role that statistical and soft condensed matter physics play in describing this system of fundamental biological importance, specifically reviewing classic and more modern tools from the theory of polymers. We also discuss some attempts toward unifying interpretations of the current results, pointing to possible directions for future investigation
Gene silencing and large-scale domain structure of the E. coli genome
The H-NS chromosome-organizing protein in E. coli can stabilize genomic DNA
loops, and form oligomeric structures connected to repression of gene
expression. Motivated by the link between chromosome organization, protein
binding and gene expression, we analyzed publicly available genomic data sets
of various origins, from genome-wide protein binding profiles to evolutionary
information, exploring the connections between chromosomal organization,
genesilencing, pseudo-gene localization and horizontal gene transfer. We report
the existence of transcriptionally silent contiguous areas corresponding to
large regions of H-NS protein binding along the genome, their position
indicates a possible relationship with the known large-scale features of
chromosome organization
DNA supercoiling in bacteria: state of play and challenges from a viewpoint of physics based modeling
DNA supercoiling is central to fundamental processes of living organisms. Its
average level along the chromosome and over time reflects the dynamic
equilibrium of opposite activities of topoisomerases, which are required to
relax mechanical stresses that are inevitably produced during DNA replication
and gene transcription. Supercoiling affects all scales of the spatio-temporal
organization of bacterial DNA, from the base pair to the large scale chromosome
conformation. Highlighted in vitro and in vivo in the 1960s and 1970s,
respectively, the first physical models were proposed concomitantly in order to
predict the deformation properties of the double helix. About fifteen years
later, polymer physics models demonstrated on larger scales the plectonemic
nature and the tree-like organization of supercoiled DNA. Since then, many
works have tried to establish a better understanding of the multiple
structuring and physiological properties of bacterial DNA in thermodynamic
equilibrium and far from equilibrium.
The purpose of this essay is to address upcoming challenges by thoroughly
exploring the relevance, predictive capacity, and limitations of current
physical models, with a specific focus on structural properties beyond the
scale of the double helix. We discuss more particularly the problem of DNA
conformations, the interplay between DNA supercoiling with gene transcription
and DNA replication, its role on nucleoid formation and, finally, the problem
of scaling up models. Our primary objective is to foster increased
collaboration between physicists and biologists. To achieve this, we have
reduced the respective jargon to a minimum and we provide some explanatory
background material for the two communities.Comment: 11 figure
Requirements for DNA-bridging proteins to act as topological barriers of the bacterial genome
Bacterial genomes have been shown to be partitioned into several kilobases
long chromosomal domains that are topologically independent from each other,
meaning that change of DNA superhelicity in one domain does not propagate to
neighbors. Both in vivo and in vitro experiments have been performed to
question the nature of the topological barriers at play, leading to several
predictions on possible molecular actors. Here, we address the question of
topological barriers using polymer models of supercoiled DNA chains. More
specifically, we determine under which conditions DNA-bridging proteins may act
as topological barriers. To this end, we developed a coarse-grained
bead-and-spring model and investigated its properties through Brownian dynamics
simulations. As a result, we find that DNA-bridging proteins must exert rather
strong constraints on their binding sites: they must block the diffusion of the
excess of twist through the two binding sites on the DNA molecule and,
simultaneously, prevent the rotation of one DNA segment relative to the other
one. Importantly, not all DNA-bridging proteins satisfy this second condition.
For example, single bridges formed by proteins that bind DNA non-specifically,
like H-NS dimers, are expected to fail with this respect. Our findings might
also explain, in the case of specific DNA-bridging proteins like LacI, why
multiple bridges are required to create stable independent topological domains.
Strikingly, when the relative rotation of the DNA segments is not prevented,
relaxation results in complex intrication of the two domains. Moreover, while
the value of the torsional stress in each domain may vary, their differential
is preserved. Our work also predicts that nucleoid associated proteins known to
wrap DNA must form higher protein-DNA complexes to efficiently work as
topological barriers.Comment: Accepted for publication in the Biophysical Journa
Cytosolic Crowding Drives the Dynamics of Both Genome and Cytosol in Escherichia coli Challenged with Sub-lethal Antibiotic Treatments.
In contrast to their molecular mode of action, the system-level effect of antibiotics on cells is only beginning to be quantified. Molecular crowding is expected to be a relevant global regulator, which we explore here through the dynamic response phenotypes in Escherichia coli, at single-cell resolution, under sub-lethal regimes of different classes of clinically relevant antibiotics, acting at very different levels in the cell. We measure chromosomal mobility through tracking of fast (<15Â s timescale) fluctuations of fluorescently tagged chromosomal loci, and we probe the fluidity of the cytoplasm by tracking cytosolic aggregates. Measuring cellular density, we show how the overall levels of macromolecular crowding affect both quantities, regardless of antibiotic-specific effects. The dominant trend is a strong correlation between the effects in different parts of the chromosome and between the chromosome and cytosol, supporting the concept of an overall global role of molecular crowding in cellular physiology.UKRI grant EP/T002778/
Cell boundary confinement sets the size and position of the E. coli chromosome
Although the spatiotemporal structure of the genome is crucial to its biological function, many basic questions remain unanswered on the morphology and segregation of chromosomes. Here, we experimentally show in Escherichia coli that spatial confinement plays a dominant role in determining both the chromosome size and position. In non-dividing cells with lengths increased to 10 times normal, single chromosomes are observed to expand > 4-fold in size. Chromosomes show pronounced internal dynamics but exhibit a robust positioning where single nucleoids reside robustly at mid-cell, whereas two nucleoids self-organize at 1/4 and 3/4 positions. The cell-size-dependent expansion of the nucleoid is only modestly influenced by deletions of nucleoid-associated proteins, whereas osmotic manipulation experiments reveal a prominent role of molecular crowding. Molecular dynamics simulations with model chromosomes and crowders recapitulate the observed phenomena and highlight the role of entropic effects caused by confinement and molecular crowding in the spatial organization of the chromosome
Bacterial Nucleoid: Interplay of DNA Demixing and Supercoiling
International audienceRunning title: DNA demixing and supercoiling. Abstract: This work addresses the question of the interplay of DNA demixing and supercoiling in bacterial cells. Demixing of DNA from other globular macromolecules results from the overall repulsion between all components of the system and leads to the formation of the nucleoid, which is the region of the cell that contains the genomic DNA in a rather compact form. Supercoiling describes the coiling of the axis of the DNA double helix to accommodate the torsional stress injected in the molecule by topoisomerases. Supercoiling is able to induce some compaction of the bacterial DNA, although to a lesser extent than demixing. In this paper, we investigate the interplay of these two mechanisms, with the goal of determining whether the total compaction ratio of the DNA is the mere sum or some more complex function of the compaction ratios due to each mechanism. To this end, we developed a coarse-grained bead-and-spring model and investigated its properties through Brownian dynamics simulations. This work reveals that there actually exist different regimes, depending on the crowder volume ratio and the DNA superhelical density. In particular, a regime where the effects of DNA demixing and supercoiling on the compaction of the DNA coil simply add up is shown to exist up to moderate values of the superhelical density. In contrast, the mean radius of the DNA coil no longer decreases above this threshold and may even increase again for sufficiently large crowder concentrations. Finally, the model predicts that the DNA coil may depart from the spherical geometry very close to the jamming threshold, as a trade-off between the need to minimize both the bending energy of the stiff plectonemes and the volume of the DNA coil to accommodate demixin
Preferential Localization of the Bacterial Nucleoid
International audienceProkaryotes do not make use of a nucleus membrane to segregate their genetic material from the cytoplasm, so that their nucleoid is potentially free to explore the whole volume of the cell. Nonetheless, high resolution images of bacteria with very compact nucleoids show that such spherical nucleoids are invariably positioned at the center of mononucleoid cells. The present work aims to determine whether such preferential localization results from generic (entropic) interactions between the nucleoid and the cell membrane or instead requires some specific mechanism, like the tethering of DNA at mid-cell or periodic fluctuations of the concentration gradient of given chemical species. To this end, we performed numerical simulations using a coarse-grained model based on the assumption that the formation of the nucleoid results from a segregative phase separation mechanism driven by the de-mixing of the DNA and non-binding globular macromolecules. These simulations show that the abrupt compaction of the DNA coil, which takes place at large crowder density, close to the jamming threshold, is accompanied by the re-localization of the DNA coil close to the regions of the bounding wall with the largest curvature, like the hemispherical caps of rod-like cells, as if the DNA coil were suddenly acquiring the localization properties of a solid sphere. This work therefore supports the hypothesis that the localization of compact nucleoids at regular cell positions involves either some anchoring of the DNA to the cell membrane or some dynamical localization mechanism
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