Visualization of living E. coli nucleoids, defined by HupA-mCherry, reveals a discrete, dynamic helical ellipsoid. Three basic features emerge. (i) Nucleoid density efficiently coalesces into longitudinal bundles, giving a stiff, low DNA density ellipsoid. (ii) This ellipsoid is radially confined within the cell cylinder. Radial confinement gives helical shape and drives and directs global nucleoid dynamics, including sister segregation. (iii) Longitudinal density waves flux back and forth along the nucleoid, with 5–10% of density shifting within 5s, enhancing internal nucleoid mobility. Furthermore, sisters separate end-to-end in sequential discontinuous pulses, each elongating the nucleoid by 5–15%. Pulses occur at 20min intervals, at defined cell cycle times. This progression is mediated by sequential installation and release of programmed tethers, implying cyclic accumulation and relief of intra-nucleoid mechanical stress. These effects could comprise a chromosome-based cell cycle engine. Overall, the presented results suggest a general conceptual framework for bacterial nucleoid morphogenesis and dynamics.

Living cells proliferate by completing and coordinating two cycles, a division cycle controlling cell size and a DNA replication cycle controlling the number of chromosomal copies. It remains unclear how bacteria such as Escherichia coli tightly coordinate those two cycles across a wide range of growth conditions. Here, we used time-lapse microscopy in combination with microfluidics to measure growth, division and replication in single E. coli cells in both slow and fast growth conditions. To compare different phenomenological cell cycle models, we introduce a statistical framework assessing their ability to capture the correlation structure observed in the data. In combination with stochastic simulations, our data indicate that the cell cycle is driven from one initiation event to the next rather than from birth to division and is controlled by two adder mechanisms: the added volume since the last initiation event determines the timing of both the next division and replication initiation events.

SUMMARY Mammalian mitotic chromosome morphogenesis was analyzed by 4D live cell and snapshot deconvolution fluorescence imaging. Prophase chromosomes, whose organization was previously unknown, are revealed to comprise co-oriented sister linear loop arrays displayed along a single, peripheral, regularly-kinked Topoisomerase II/cohesin/condensin II axis. Thereafter, rather than smooth, progressive compaction as generally envisioned, progression to metaphase is a discontinuous process involving chromosome expansion as well as compaction. At Late Prophase, dependent on Topoisomerase II and with concomitant cohesin release, chromosomes expand, axes split and straighten, and chromatin loops transit to a radial disposition around now-central axes. Finally, chromosomes globally compact, giving the metaphase state. These patterns are consistent with the hypothesis that the molecular events of chromosome morphogenesis are governed by accumulation and release of chromosome stress, created by chromatin compaction and expansion. Chromosome state could evolve analogously throughout the cell cycle.

Atomic force microscopy was used to image singlestranded DNA (ssDNA) adsorbed on mica modified by Mg 2+ , by 3-aminopropyltriethoxysilane or on modified highly oriented pyrolytic graphite (HOPG). ssDNA molecules on mica have compact structures with lumps, loops and super twisting, while on modified HOPG graphite ssDNA molecules adopt a conformation without secondary structures. We have shown that the immobilization of ssDNA under standard conditions on modified HOPG eliminates intramolecular base-pairing, thus this method could be important for studying certain processes involving ssDNA in more details.

Living cells proliferate by completing and coordinating two essential cycles, a division 9 cycle that controls cell size, and a DNA replication cycle that controls the number of chromosomal 10 copies in the cell. Despite lacking dedicated cell cycle control regulators such as cyclins in 11 eukaryotes, bacteria such as E. coli manage to tightly coordinate those two cycles across a wide 12 range of growth conditions, including situations where multiple nested rounds of replication 13 progress simultaneously. Various cell cycle models have been proposed to explain this feat, but it 14 has been impossible to validate them so far due to a lack of experimental tools for systematically 15 testing their different predictions. Recently new insights have been gained on the division cycle 16 through the study of the structure of fluctuations in growth, size, and division in individual cells. In 17 particular, it was found that cell size appears to be controlled by an adder mechanism, i.e. the 18 added volume between divisions is held approximately constant and fluctuates independently of 19 growth rate and cell size at birth. However, how replication initiation is regulated and coupled to 20 cell size control remains unclear, mainly due to scarcity of experimental measurements on 21 replication initiation at the single-cell level. Here, we used time-lapse microscopy in combination 22 with microfluidics to directly measure growth, division and replication in thousands of single E. coli 23 cells growing in both slow and fast growth conditions. In order to compare different 24 phenomenological models of the cell cycle, we introduce a statistical framework which assess their 25 ability to capture the correlation structure observed in the experimental data. Using this in 26 combination with stochastic simulations, our data indicate that, instead of thinking of the cell cycle 27 as running from birth to division, the cell cycle is controlled by two adder mechanisms starting at 28 the initiation of replication: the added volume since the last initiation event controls the timing of 29 both the next division event and the next replication initiation event. Interestingly the double-adder 30 mechanism identified in this study has recently been found to explain the more complex cell cycle 31 of mycobacteria, suggesting shared control strategies across species. 32 33 34 Across all domains of life, cell proliferation requires that the chromosome replication and cell 35 division cycles are coordinated to ensure that every new cell receives one copy of the genetic 36 material. While in eukaryotes this coordination is implemented by a dedicated regulatory system in 37which genome replication and division occur in well-separated stages, no such system has been 38 found in most bacteria. This suggests that the molecular events that control replication initiation 39 1 of 20 and division might be coordinated more directly in bacteria, through molecular interactions that 40 are yet to be elucidated. The contrast between this effi...

The scaling properties of DNA knots of different complexities were studied by atomic force microscope. Following two different protocols DNA knots are adsorbed onto a mica surface in regimes of (i) strong binding, that induces a kinetic trapping of the three-dimensional (3D) configuration, and of (ii) weak binding, that permits (partial) relaxation on the surface. In (i) the radius of gyration of the adsorbed DNA knot scales with the 3D Flory exponent 0:60 within error. In (ii), we find 0:66, a value between the 3D and 2D ( 3=4) exponents. Evidence is also presented for the localization of knot crossings in 2D under weak adsorption conditions. DOI: 10.1103/PhysRevLett.98.058102 PACS numbers: 87.64.Dz, 36.20.Ey, 82.35.Gh, 87.14.Gg The first systematic study of knots was undertaken by Tait in the 19th century [1], following Kelvin's theory of vortex atoms [2]. During the 20th century progress was made understanding knots in a topological framework and invariants were found to classify them [3,4]. Experimentally, knots remained elusive and difficult to study, but the discovery of their role in biological processes [5,6] revived the interest in their properties. For example, knots on DNA inhibit its separation into single strands during replication, impede access to the full genetic code during transcription, are implicated in gene regulation [7], and influence DNA stability [8]. Replication and transcription of circular DNA are controlled by topoisomerases [5] promoting questions on the detailed mechanism of enzymatic knot detection [9]. Finally, knots have been found in proteins in their native states [10]. The physical interest in the behavior of DNA knots concerns two main questions: (i) the scaling properties of the radius of gyration R g [11] and (ii) knot localization.(i) From simulations and scaling arguments, it is commonly accepted that the gyration radius of knots to leading order scales as R g ' AL , for all knot types, as long as the polymer is sufficiently long [12 -14], where L is the contour length. Here, we quantify the Flory exponent of 3D and 2D configurations by determining the fractal dimension of single DNA knots.(ii) From a polymer physics interest, and to understand better the action of topoisomerases and the physiological role of DNA knots, it is crucial to find out whether knots segregate into simply connected rings, with all essential crossings confined in a knot region of contour length s much smaller than the overall chain length L. Such localization has been predicted theoretically in 2D as a consequence of entropic maximization [15]. Simulations in 3D yield a size distribution of the knot region that is peaked well below L for fixed knot types [16], and the size s of the knot region scales as s L t , with t < 1 [13,14]. It is experimentally difficult to probe the predicted scaling behavior s L t , since L would have to be varied significantly. This is at present out of reach given the available techniques used to prepare the DNA knots.Here we study the scaling properties and chain ...

The conformation of circular DNA molecules of various lengths adsorbed in a 2D conformation on a mica surface is studied. The results confirm the conjecture that the critical exponent nu is topologically invariant and equal to the self-avoiding walk value (in the present case nu=3/4), and that the topology and dimensionality of the system strongly influence the crossover between the rigid regime and the self-avoiding regime at a scale L approximately 7l{p}. Additionally, the bond correlation function scales with the molecular length L as predicted. For molecular lengths L

Chromosomal and plasmid DNA molecules in bacterial cells are maintained under torsional tension and are therefore supercoiled. With the exception of extreme thermophiles, supercoiling has a negative sign, which means that the torsional tension diminishes the DNA helicity and facilitates strand separation. In consequence, negative supercoiling aids such processes as DNA replication or transcription that require global- or local-strand separation. In extreme thermophiles, DNA is positively supercoiled which protects it from thermal denaturation. While the role of DNA supercoiling connected to the control of DNA stability, is thoroughly researched and subject of many reviews, a less known role of DNA supercoiling emerges and consists of aiding DNA topoisomerases in DNA decatenation and unknotting. Although DNA catenanes are natural intermediates in the process of DNA replication of circular DNA molecules, it is necessary that they become very efficiently decatenated, as otherwise the segregation of freshly replicated DNA molecules would be blocked. DNA knots arise as by-products of topoisomerase-mediated intramolecular passages that are needed to facilitate general DNA metabolism, including DNA replication, transcription or recombination. The formed knots are, however, very harmful for cells if not removed efficiently. Here, we overview the role of DNA supercoiling in DNA unknotting and decatenation.