Bacterial Chromosome Dynamics Research Articles

Molecular Dynamics Simulation of a Feather-Boa Model of a Bacterial Chromosome.

The chromosome of a bacterium consists of a mega-base pair-long circular DNA, which self-organizes within the micron-sized bacterial cell volume, compacting itself by three orders of magnitude. Unlike eukaryotic chromosomes, it lacks a nuclear membrane and freely floats in the cytosol confined by the cell membrane. It is believed that strong confinement, cross-linking by associated proteins, and molecular crowding all contribute to determine chromosome size and morphology. Modelling the chromosome simply as a circular polymer decorated with closed side loops in a cylindrical confining volume has been shown to already recapture some of the salient properties observed experimentally. Here we describe how a computer simulation can be set up to study structure and dynamics of bacterial chromosomes using this model.

Dynamics of Bacterial Chromosomes by Locus Tracking in Fluorescence Microscopy.

In the last two decades, it has been shown that bacterial chromosomes have remarkable spatial organization at various scales, and they display well-defined movements during the cell cycle, for example to reliably segregate daughter chromosomes. More recently, various labs have begun investigating also the short time dynamics (displacements during time intervals of 0.1s-100s), which should be related to the molecular structure. Probing these dynamics is analogous to "microrheology" approaches that have been applied successfully to study mechanical response of complex fluids. These studies of chromosome fluctuation dynamics have revealed differences of fluctuation amplitude across the chromosome, and different characters of motion depending on the time window of interest. Different fluctuation amplitudes have also been observed for the same chromosomal loci under antibiotic treatments, with magnitudes that are correlated to changes in intracellular density and thus crowding. We describe how to carry out tracking experiments of single loci and how to analyze locus motility. We point out the importance of considering in the analysis the number of GFP molecules per fluorescent locus, as well as the nature of the protein they are fused to, and also how to measure intracellular density.

A Bidimensional Segregation Mode Maintains Symbiont Chromosome Orientation toward Its Host.

All living organisms require accurate segregation of their genetic material. However, in microbes, chromosome segregation is less understood than replication and cell division, which makes its decipherment a compelling research frontier. Furthermore, it has only been studied in free-living microbes so far. Here, we investigated this fundamental process in arod-shaped symbiont, Candidatus Thiosymbion oneisti. This gammaproteobacterium divides longitudinally as to form a columnar epithelium ensheathing its nematode host. We hypothesized that uninterrupted host attachment would affect bacterial chromosome dynamics and set out to localize specific chromosomal loci and putative DNA-segregating proteins by fluorescence in situ hybridization and immunostaining, respectively. First, DNA replication origins (ori) number per cell demonstrated symbiont monoploidy. Second, we showed that sister ori segregate diagonally prior to septation onset. Moreover, the localization pattern of the centromere-binding protein ParB recapitulates that of ori, and consistently, we showed recombinant ParB to specifically bind an ori-proximal site (parS) invitro. Third, chromosome replication ends prior to cell fission, and as the poles start to invaginate, termination of replication (ter) sites localize medially, at the leading edges of the growing septum. They then migrate to midcell, concomitantly with septation progression and until this is completed. In conclusion, we propose that symbiont ParB might drive chromosome segregation along the short axis and that tethering of sister ter regions to the growing septum mediates their migration along the long axis. Crucially, active bidimensional segregation of the chromosome allows transgenerational maintenance of its configuration, and therefore, it may represent an adaptation to symbiosis. VIDEO ABSTRACT.

Confinement and crowding control the morphology and dynamics of a model bacterial chromosome.

Motivated by recent experiments probing the shape, size and dynamics of bacterial chromosomes in growing cells, we consider a polymer model consisting of a circular backbone to which side-loops are attached, confined to a cylindrical cell. Such a model chromosome spontaneously adopts a helical shape, which is further compacted by molecular crowders to occupy a nucleoid-like sub-volume of the cell. With increasing cell length, the longitudinal size of the chromosome increases in a non-linear fashion until finally saturating, its morphology gradually opening up while displaying a changing number of helical turns. For shorter cells, the chromosome extension varies non-monotonically with cell size, which we show is associated with a radial to longitudinal spatial reordering of the crowders. Confinement and crowders constrain chain dynamics leading to anomalous diffusion. While the scaling exponent for the mean squared displacement of center of mass grows and saturates with cell length, that of individual loci displays a broad distribution with a sharp maximum.

Tracking Bacterial Chromosome Dynamics with Microfluidics-Based Live Cell Imaging.

In bacteria, chromosomes are highly organized within the limited volume of the cell to form a nucleoid. Recent application of microscopy and chromosome conformation capture techniques have together provided a comprehensive understanding of the nature of this organization and the role of factors such as the structural maintenance of chromosomes (SMC) proteins in the establishment and maintenance of the same. In this chapter, we outline a microfluidics-based approach for live cell imaging of Escherichia coli chromosome dynamics in wild-type cells. This assay can be used to track the activity of the SMC complex, MukBEF, on DNA and assess the impact of perturbations such as DNA damage on chromosome organization and segregation.

Molecular Dynamics Simulation of a Feather-Boa Model of a Bacterial Chromosome.

The chromosome of a bacterium consists of a mega-base pair long circular DNA, which self-organizes within the micron-sized bacterial cell volume, compacting itself by three orders of magnitude. Unlike in eukaryotes, it lacks a nuclear membrane, and freely floats in the cytosol confined by the cell membrane. It is believed that strong confinement, cross-linking by associated proteins, and molecular crowding all contribute to determine chromosome size and morphology. Modeling the chromosome simply as a circular polymer decorated with closed side-loops in a cylindrical confining volume, has been shown to already recapture some of the salient properties observed experimentally. Here, we describe how a computer simulation can be set up to study structure and dynamics of bacterial chromosomes using this model.

Bacterial Chromosome Dynamics by Locus Tracking in Fluorescence Microscopy.

Bacterial chromosomes have been shown in the last two decades to have remarkable spatial organization at various scales, and also well-defined movements during the cell cycle, for example, to reliably segregate daughter chromosomes. More recently, various labs have begun investigating the short-time dynamics (displacements during time intervals of 0.1-100 s), which one hopes to link to structure, in analogy to "microrheology" approaches applied successfully to study mechanical response of complex fluids. These studies of chromosome fluctuation dynamics have revealed differences of fluctuation amplitude across the chromosome, and different characters of motion depending on the time window of interest. The highly nontrivial motion at the shortest experimentally accessible times is still not fully understood in terms of physical models of DNA and cytosol. We describe how to carry out tracking experiments of single locus and how to analyze locus motility. We point out the importance of considering in the analysis the number of GFP molecules per fluorescent locus.

Resolving Chromosome Segregation in Bacteria

Bacterial chromosomes are evenly distributed between daughter cells, however no equivalent eukaryotic mitotic apparatus has been identified yet. Nevertheless, an advance in our understanding of the dynamics of the bacterial chromosome has been accomplished in recent years by adopting fluorescence microscopy techniques to visualize living bacterial cells. Here, some of the most recent studies that yield new insights into the nature of bacterial chromosome dynamics are described. In addition, we review in detail the current models that attempt to illuminate the mechanism of chromosome segregation in bacteria and discuss the possibility that a bacterial mitotic apparatus does indeed exist.

Measuring chromosome dynamics on different time scales using resolvases with varying half-lives.

The bacterial chromosome is organized into multiple independent domains, each capable of constraining the plectonemic negative supercoil energy introduced by DNA gyrase. Different experimental approaches have estimated the number of domains to be between 40 and 150. The site-specific resolution systems of closely related transposons Tn3 and gammadelta are valuable tools for measuring supercoil diffusion and analysing bacterial chromosome dynamics in vivo. Once made, the wild-type resolvase persists in cells for time periods greater than the cell doubling time. To examine chromosome dynamics over shorter time frames that are more closely tuned to processes like inducible transcription, we constructed a set of resolvases with cellular half-lives ranging from less than 5 min to 30 min. Analysing chromosomes on different time scales shows domain structure to be dynamic. Rather than the 150 domains detected with the Tn3 resolvase, wild-type cells measured over a 10 min time span have more than 400 domains per genome equivalent, and some gyrase mutants exceed 1000.

Bacterial chromosome dynamics.

Bacterial chromosomes are highly compacted structures and share many properties with their eukaryote counterparts, despite not being organized into chromatin or being contained within a cell nucleus. Proteins conserved across all branches of life act in chromosome organization, and common mechanisms maintain genome integrity and ensure faithful replication. The principles that underlie chromosome segregation in bacteria and eukaryotes share similarities, although bacteria segregate DNA as it replicates and lack a eukaryote-like mitotic apparatus for segregating chromosomes. This may be because the distances that newly replicated bacterial chromosomes move apart before cell division are small as compared to those in eukaryotes. Bacteria specify positional information, which determines where cell division will occur and which places the replication machinery and chromosomal loci at defined locations that change during cell cycle progression.

Chromosomes Through Space and Time

Bacterial chromosomes, because of the much smaller size of prokaryotic cells, have traditionally been analyzed by genetic rather than cytological means. Indeed, Kohiyama et al. (p. 802) celebrate the 50th anniversary of the discovery of bacterial conjugation (or “sex”), which has been the foundation of bacterial genetics; revealing, for example, that bacterial chromosomes are circular, rather than linear as in eukaryotes. Contents Reviews and Viewpoints Bacterial Chromosome Dynamics D. J. Sherratt [Chromosome Choreography: The Meiotic Ballet][1] S. L. Page and R. S. Hawley Prokaryotic Chromosomes and Disease J. Hacker et al. Structual Dynamics of Eukaryotic Chromosome Evolution E. E. Eichler and D. Sankoff [Heterochromatin and Epigenetic Control of Gene Expression][2] S. I. S. Grewal and D. Moazed Bacterial Sex: Playing Voyeurs 50 Years Later M. Kohiyama et al. See also related material on [Science's STKE][3]. E ukaryotic chromosomes were first (unwittingly) observed over 100 years ago because of their highly dynamic nature during cell division. Understanding their sheer physicality—the spectacular events that result in the segregation of a complete set of chromosomes to two daughter cells—has been a major driving force in chromosome research ever since. The fact that they also happen to harbor the genome has added (among other elements) the dimension of evolutionary time to their study. Nevertheless, bacteria, too, have a complex and dynamic chromosome segregation process that, like that of eukaryotes, must be able to cope with multiple chromosomes. But here the multiple chromosomes are all copies of the single circular genomic DNA, which continues to be replicated during the act of segregation (Sherratt, p. [780][4]). Despite these differences, many proteins guiding the dynamics of bacterial chromosomes (such as their replication, recombination, and repair) have evolutionarily related counterparts in eukaryotes. Unlike bacterial sex, sex in eukaryotes—at least in terms of chromosome dynamics during meiosis, the specialized cell division required to generate germ cells—is of necessity a highly regimented affair. Ensuring the correct segregation of chromosomes to the daughter cells has required the evolution of a vastly complex segregation machinery that is only starting to yield up its secrets, as discussed by Page and Hawley (p. 785). As well as being highly dynamic within the cell, the structure of the chromosomes and the genome has varied widely across evolutionary time, driven in part by the recombination intermediates often required for chromosome alignment during meiosis. This dynamism has had a profound effect on the course of evolution of both prokaryotes and eukaryotes. Perhaps the greatest impact of the evolutionary variability of prokaryotic chromosomes on humans and human history has been through infectious disease (Hacker et al. , p. 790). Eukaryotic chromosomes have also seen tremendous flux over the course of evolution, with the malleability of the genome often being concentrated at “hotspots” at the centromeres, telomeres, and other repeated sequences but also including chromosome and entire genome duplications (Eichler and Sankoff, p. [793][5]). But what of the constituents of chromosomes? In eukaryotes, histones are the principal protein components of chromatin. Although histones were once thought of as little more than inert packaging material, it is now clear that covalent histone modifications are crucial for the regulation of gene expression through the formation of refractile heterochromatin and permissive euchromatin (Grewal and Moazed, p. 798). Surprisingly, noncoding RNA is also involved in the formation of centromeric heterochromatin, which is essential for the chromosome segregation first seen over 100 years ago. [1]: /lookup/doi/10.1126/science.1086605 [2]: /lookup/doi/10.1126/science.1086887 [3]: http://stke.sciencemag.org/ [4]: /lookup/doi/10.1126/science.1084780 [5]: /lookup/doi/10.1126/science.1086132