A single fiber view of the nucleosome organization in eukaryotic chromatin.

Eukaryotic cells are thought to arrange nucleosomes into extended arrays with evenly spaced nucleosomes phased at genomic landmarks. Here we tested to what extent this stereotypic organization describes the nucleosome organization in Saccharomyces cerevisiae using Fiber-Seq, a long-read sequencing technique that maps entire nucleosome arrays on individual chromatin fibers in a high throughput manner. With each fiber coming from a different cell, Fiber-Seq uncovers cell-to-cell heterogeneity. The long reads reveal the nucleosome architecture even over repetitive DNA such as the ribosomal DNA repeats. The absolute nucleosome occupancy, a parameter that is difficult to obtain with conventional sequencing approaches, is a direct readout of Fiber-Seq. We document substantial deviations from the stereotypical nucleosome organization with unexpectedly long linker DNAs between nucleosomes, gene bodies missing entire nucleosomes, cell-to-cell heterogeneity in nucleosome occupancy, heterogeneous phasing of arrays and irregular nucleosome spacing. Nucleosome array structures are indistinguishable throughout the gene body and with respect to the direction of transcription arguing against transcription promoting array formation. Acute nucleosome depletion destroyed most of the array organization indicating that nucleosome remodelers cannot efficiently pack nucleosomes under those conditions. Given that nucleosomes are cis-regulatory elements, the cell-to-cell heterogeneity uncovered by Fiber-Seq provides much needed information to understand chromatin structure and function.

From Brownian to Deterministic Motor Movement in a DNA-Based Molecular Rotor.

Molecular devices that have an anisotropic periodic potential landscape can be operated as Brownian motors. When the potential landscape is cyclically switched with an external force, such devices can harness random Brownian fluctuations to generate a directed motion. Recently, directed Brownian motor-like rotatory movement was demonstrated with an electrically switched DNA origami rotor with designed ratchet-like obstacles. Here, we demonstrate that the intrinsic anisotropy of DNA origami rotors is also sufficient to result in motor movement. We show that for low amplitudes of an external switching field, such devices operate as Brownian motors, while at higher amplitudes, they behave deterministically as overdamped electrical motors. We characterize the amplitude and frequency dependence of the movements, showing that after an initial steep rise, the angular speed peaks and drops for excessive driving amplitudes and frequencies. The rotor movement can be well described by a simple stochastic model of the system.

Pressure-induced shape-shifting of helical bacteria.

Many bacterial species are helical in shape, including the widespread pathogen H. pylori. Motivated by recent experiments on H. pylori showing that cell wall synthesis is not uniform [J. A. Taylor, et al., eLife, 2020, 9, e52482], we investigate the possible formation of helical cell shape induced by elastic heterogeneity. We show, experimentally and theoretically, that helical morphogenesis can be produced by pressurizing an elastic cylindrical vessel with helical reinforced lines. The properties of the pressurized helix are highly dependent on the initial helical angle of the reinforced region. We find that steep angles result in crooked helices with, surprisingly, a reduced end-to-end distance upon pressurization. This work helps explain the possible mechanisms for the generation of helical cell morphologies and may inspire the design of novel pressure-controlled helical actuators.

A global resource allocation strategy governs growth transition kinetics of Escherichia coli.

A grand challenge of systems biology is to predict the kinetic responses of living systems to perturbations starting from the underlying molecular interactions. Changes in the nutrient environment have long been used to study regulation and adaptation phenomena in microorganisms and they remain a topic of active investigation. Although much is known about the molecular interactions that govern the regulation of key metabolic processes in response to applied perturbations, they are insufficiently quantified for predictive bottom-up modelling. Here we develop a top-down approach, expanding the recently established coarse-grained proteome allocation models from steady-state growth into the kinetic regime. Using only qualitative knowledge of the underlying regulatory processes and imposing the condition of flux balance, we derive a quantitative model of bacterial growth transitions that is independent of inaccessible kinetic parameters. The resulting flux-controlled regulation model accurately predicts the time course of gene expression and biomass accumulation in response to carbon upshifts and downshifts (for example, diauxic shifts) without adjustable parameters. As predicted by the model and validated by quantitative proteomics, cells exhibit suboptimal recovery kinetics in response to nutrient shifts owing to a rigid strategy of protein synthesis allocation, which is not directed towards alleviating specific metabolic bottlenecks. Our approach does not rely on kinetic parameters, and therefore points to a theoretical framework for describing a broad range of such kinetic processes without detailed knowledge of the underlying biochemical reactions.

Protein-protein interaction network controlling establishment and maintenance of switchable cell polarity.

Cell polarity underlies key processes in all cells, including growth, differentiation and division. In the bacterium Myxococcus xanthus, front-rear polarity is crucial for motility. Notably, this polarity can be inverted, independent of the cell-cycle, by chemotactic signaling. However, a precise understanding of the protein network that establishes polarity and allows for its inversion has remained elusive. Here, we use a combination of quantitative experiments and data-driven theory to unravel the complex interplay between the three key components of the M. xanthus polarity module. By studying each of these components in isolation and their effects as we systematically reconstruct the system, we deduce the network of effective interactions between the polarity proteins. RomR lies at the root of this network, promoting polar localization of the other components, while polarity arises from interconnected negative and positive feedbacks mediated by the small GTPase MglA and its cognate GAP MglB, respectively. We rationalize this network topology as operating as a spatial toggle switch, providing stable polarity for persistent cell movement whilst remaining responsive to chemotactic signaling and thus capable of polarity inversions. Our results have implications not only for the understanding of polarity and motility in M. xanthus but also, more broadly, for dynamic cell polarity.

Absolute nucleosome occupancy map for the Saccharomyces cerevisiae genome.

Mapping of nucleosomes, the basic DNA packaging unit in eukaryotes, is fundamental for understanding genome regulation because nucleosomes modulate DNA access by their positioning along the genome. A cell-population nucleosome map requires two observables: nucleosome positions along the DNA ("Where?") and nucleosome occupancies across the population ("In how many cells?"). All available genome-wide nucleosome mapping techniques are yield methods because they score either nucleosomal (e.g., MNase-seq, chemical cleavage-seq) or nonnucleosomal (e.g., ATAC-seq) DNA but lose track of the total DNA population for each genomic region. Therefore, they only provide nucleosome positions and maybe compare relative occupancies between positions, but cannot measure absolute nucleosome occupancy, which is the fraction of all DNA molecules occupied at a given position and time by a nucleosome. Here, we established two orthogonal and thereby cross-validating approaches to measure absolute nucleosome occupancy across the Saccharomyces cerevisiae genome via restriction enzymes and DNA methyltransferases. The resulting high-resolution (9-bp) map shows uniform absolute occupancies. Most nucleosome positions are occupied in most cells: 97% of all nucleosomes called by chemical cleavage-seq have a mean absolute occupancy of 90 ± 6% (±SD). Depending on nucleosome position calling procedures, there are 57,000 to 60,000 nucleosomes per yeast cell. The few low absolute occupancy nucleosomes do not correlate with highly transcribed gene bodies, but correlate with increased presence of the nucleosome-evicting chromatin structure remodeling (RSC) complex, and are enriched upstream of highly transcribed or regulated genes. Our work provides a quantitative method and reference frame in absolute terms for future chromatin studies.

Emergence of Colloidal Patterns in ac Electric Fields.

Suspended microparticles subjected to ac electrical fields collectively organize into band patterns perpendicular to the field direction. The bands further develop into zigzag shaped patterns, in which the particles are observed to circulate. We demonstrate that this phenomenon can be observed quite generically by generating such patterns with a wide range of particles: silica spheres, fatty acid, oil, and coacervate droplets, bacteria, and ground coffee. We show that the phenomenon can be well understood in terms of second order electrokinetic flow, which correctly predicts the hydrodynamic interactions required for the pattern formation process. Brownian particle simulations based on these interactions accurately recapitulate all of the observed pattern formation and symmetry-breaking events, starting from a homogeneous particle suspension. The emergence of the formed patterns can be predicted quantitatively within a parameter-free theory.

Four different mechanisms for switching cell polarity.

The mechanisms and design principles of regulatory systems establishing stable polarized protein patterns within cells are well studied. However, cells can also dynamically control their cell polarity. Here, we ask how an upstream signaling system can switch the orientation of a polarized pattern. We use a mathematical model of a core polarity system based on three proteins as the basis to study different mechanisms of signal-induced polarity switching. The analysis of this model reveals four general classes of switching mechanisms with qualitatively distinct behaviors: the transient oscillator switch, the reset switch, the prime-release switch, and the push switch. Each of these regulatory mechanisms effectively implements the function of a spatial toggle switch, however with different characteristics in their nonlinear and stochastic dynamics. We identify these characteristics and also discuss experimental signatures of each type of switching mechanism.

Dynamics of chromosomal target search by a membrane-integrated one-component receptor.

Membrane proteins account for about one third of the cellular proteome, but it is still unclear how dynamic they are and how they establish functional contacts with cytoplasmic interaction partners. Here, we consider a membrane-integrated one-component receptor that also acts as a transcriptional activator, and analyze how it kinetically locates its specific binding site on the genome. We focus on the case of CadC, the pH receptor of the acid stress response Cad system in E. coli. CadC is a prime example of a one-component signaling protein that directly binds to its cognate target site on the chromosome to regulate transcription. We combined fluorescence microscopy experiments, mathematical analysis, and kinetic Monte Carlo simulations to probe this target search process. Using fluorescently labeled CadC, we measured the time from activation of the receptor until successful binding to the DNA in single cells, exploiting that stable receptor-DNA complexes are visible as fluorescent spots. Our experimental data indicate that CadC is highly mobile in the membrane and finds its target by a 2D diffusion and capture mechanism. DNA mobility is constrained due to the overall chromosome organization, but a labeled DNA locus in the vicinity of the target site appears sufficiently mobile to randomly come close to the membrane. Relocation of the DNA target site to a distant position on the chromosome had almost no effect on the mean search time, which was between four and five minutes in either case. However, a mutant strain with two binding sites displayed a mean search time that was reduced by about a factor of two. This behavior is consistent with simulations of a coarse-grained lattice model for the coupled dynamics of DNA within a cell volume and proteins on its surface. The model also rationalizes the experimentally determined distribution of search times. Overall our findings reveal that DNA target search does not present a much bigger kinetic challenge for membrane-integrated proteins than for cytoplasmic proteins. More generally, diffusion and capture mechanisms may be sufficient for bacterial membrane proteins to establish functional contacts with cytoplasmic targets.

Enzyme-Free Copying of 12 Bases of RNA with Dinucleotides.

The synthesis of complementary strands is the reaction underlying the replication of genetic information. It is likely that the earliest self-replicating systems used RNA as genetic material. How RNA was copied in the absence of enzymes and what sequences were most likely to have supported replication is not clear. Here we show that mixtures of dinucleotides with C and G as bases copy an RNA sequence of up to 12 nucleotides in dilute aqueous solution. Successful enzyme-free copying occurred with in situ activation at 4 °C and pH 6.0. Dimers were incorporated in favor of monomers when both competed as reactants, and little misincorporation was detectable in mass spectra. Simulations using experimental rate constants confirmed that mixed C/G sequences are good candidates for successful replication with dimers. Because dimers are intermediates in the synthesis of longer strands, our results support evolutionary scenarios encompassing formation and copying of RNA strands in enzyme-free fashion.

Slower growth of Escherichia coli leads to longer survival in carbon starvation due to a decrease in the maintenance rate.

Fitness of bacteria is determined both by how fast cells grow when nutrients are abundant and by how well they survive when conditions worsen. Here, we study how prior growth conditions affect the death rate of Escherichia coli during carbon starvation. We control the growth rate prior to starvation either via the carbon source or via a carbon-limited chemostat. We find a consistent dependence where death rate depends on the prior growth conditions only via the growth rate, with slower growth leading to exponentially slower death. Breaking down the observed death rate into two factors, maintenance rate and recycling yield, reveals that slower growing cells display a decreased maintenance rate per cell volume during starvation, thereby decreasing their death rate. In contrast, the ability to scavenge nutrients from carcasses of dead cells (recycling yield) remains constant. Our results suggest a physiological trade-off between rapid proliferation and long survival. We explore the implications of this trade-off within a mathematical model, which can rationalize the observation that bacteria outside of lab environments are not optimized for fast growth.

Dynamics of the Buckling Transition in Double-Stranded DNA and RNA.

DNA under torsional strain undergoes a buckling transition that is the fundamental step in plectoneme nucleation and supercoil dynamics, which are critical for the processing of genomic information. Despite its importance, quantitative models of the buckling transition, in particular to also explain the surprising two-orders-of-magnitude difference between the buckling times for RNA and DNA revealed by single-molecule tweezers experiments, are currently lacking. Additionally, little is known about the configurations of the DNA during the buckling transition because they are not directly observable experimentally. Here, we use a discrete worm-like chain model and Brownian dynamics to simulate the DNA/RNA buckling transition. Our simulations are in good agreement with experimentally determined parameters of the buckling transition. The simulations show that the buckling time strongly and exponentially depends on the bending stiffness, which accounts for more than half the measured difference between DNA and RNA. Analyzing the microscopic conformations of the chain revealed by our simulations, we find clear evidence for a solenoid-shaped transition state and a curl intermediate. The curl intermediate features a single loop and becomes increasingly populated at low forces. Taken together, the simulations suggest that the worm-like chain model can account semiquantitatively for the buckling dynamics of both DNA and RNA.

Optimal Compartmentalization Strategies for Metabolic Microcompartments.

Intracellular compartmentalization of cooperating enzymes is a strategy that is frequently used by cells. Segregation of enzymes that catalyze sequential reactions can alleviate challenges such as toxic pathway intermediates, competing metabolic reactions, and slow reaction rates. Inspired by nature, synthetic biologists also seek to encapsulate engineered metabolic pathways within vesicles or proteinaceous shells to enhance the yield of industrially and pharmaceutically useful products. Although enzymatic compartments have been extensively studied experimentally, a quantitative understanding of the underlying design principles is still lacking. Here, we study theoretically how the size and enzymatic composition of compartments should be chosen so as to maximize the productivity of a model metabolic pathway. We find that maximizing productivity requires compartments larger than a certain critical size. The enzyme density within each compartment should be tuned according to a power-law scaling in the compartment size. We explain these observations using an analytically solvable, well-mixed approximation. We also investigate the qualitatively different compartmentalization strategies that emerge in parameter regimes where this approximation breaks down. Our results suggest that the different sizes and enzyme packings of α- and ß-carboxysomes each constitute an optimal compartmentalization strategy given the properties of their respective protein shells.

Single-cell characterization of metabolic switching in the sugar phosphotransferase system of Escherichia coli.

The utilization of several sugars in Escherichia coli is regulated by the Phosphotransferase System (PTS), in which diverse sugar utilization modules compete for phosphoryl flux from the general PTS proteins. Existing theoretical work predicts a winner-take-all outcome when this flux limits carbon uptake. To date, no experimental work has interrogated competing PTS uptake modules with single-cell resolution. Using time-lapse microscopy in perfused microchannels, we analyzed the competition between N-acetyl-glucosamine and sorbitol, as representative PTS sugars, by measuring both the expression of their utilization systems and the concomitant impact of sugar utilization on growth rates. We find two distinct regimes: hierarchical usage of the carbohydrates, and co-expression of the genes for both systems. Simulations of a mathematical model incorporating asymmetric sugar quality reproduce our metabolic phase diagram, indicating that under conditions of nonlimiting phosphate flux, co-expression is due to uncoupling of both sugar utilization systems. Our model reproduces hierarchical winner-take-all behaviour and stochastic co-expression, and predicts the switching between both strategies as a function of available phosphate flux. Hence, experiments and theory both suggest that PTS sugar utilization involves not only switching between the sugars utilized but also switching of utilization strategies to accommodate prevailing environmental conditions.

The efficiency of driving chemical reactions by a physical non-equilibrium is kinetically controlled.

An out-of-equilibrium physical environment can drive chemical reactions into thermodynamically unfavorable regimes. Under prebiotic conditions such a coupling between physical and chemical non-equilibria may have enabled the spontaneous emergence of primitive evolutionary processes. Here, we study the coupling efficiency within a theoretical model that is inspired by recent laboratory experiments, but focuses on generic effects arising whenever reactant and product molecules have different transport coefficients in a flow-through system. In our model, the physical non-equilibrium is represented by a drift-diffusion process, which is a valid coarse-grained description for the interplay between thermophoresis and convection, as well as for many other molecular transport processes. As a simple chemical reaction, we consider a reversible dimerization process, which is coupled to the transport process by different drift velocities for monomers and dimers. Within this minimal model, the coupling efficiency between the non-equilibrium transport process and the chemical reaction can be analyzed in all parameter regimes. The analysis shows that the efficiency depends strongly on the Damköhler number, a parameter that measures the relative timescales associated with the transport and reaction kinetics. Our model and results will be useful for a better understanding of the conditions for which non-equilibrium environments can provide a significant driving force for chemical reactions in a prebiotic setting.

Replication-guided nucleosome packing and nucleosome breathing expedite the formation of dense arrays.

The first level of genome packaging in eukaryotic cells involves the formation of dense nucleosome arrays, with DNA coverage near 90% in yeasts. How cells achieve such high coverage within a short time, e.g. after DNA replication, remains poorly understood. It is known that random sequential adsorption of impenetrable particles on a line reaches high density extremely slowly, due to a jamming phenomenon. The nucleosome-shifting action of remodeling enzymes has been proposed as a mechanism to resolve such jams. Here, we suggest two biophysical mechanisms which assist rapid filling of DNA with nucleosomes, and we quantitatively characterize these mechanisms within mathematical models. First, we show that the 'softness' of nucleosomes, due to nucleosome breathing and stepwise nucleosome assembly, significantly alters the filling behavior, speeding up the process relative to 'hard' particles with fixed, mutually exclusive DNA footprints. Second, we explore model scenarios in which the progression of the replication fork could eliminate nucleosome jamming, either by rapid filling in its wake or via memory of the parental nucleosome positions. Taken together, our results suggest that biophysical effects promote rapid nucleosome filling, making the reassembly of densely packed nucleosomes after DNA replication a simpler task for cells than was previously thought.

Escalation of polymerization in a thermal gradient.

For the emergence of early life, the formation of biopolymers such as RNA is essential. However, the addition of nucleotide monomers to existing oligonucleotides requires millimolar concentrations. Even in such optimistic settings, no polymerization of RNA longer than about 20 bases could be demonstrated. How then could self-replicating ribozymes appear, for which recent experiments suggest a minimal length of 200 nt? Here, we demonstrate a mechanism to bridge this gap: the escalated polymerization of nucleotides by a spatially confined thermal gradient. The gradient accumulates monomers by thermophoresis and convection while retaining longer polymers exponentially better. Polymerization and accumulation become mutually self-enhancing and result in a hyperexponential escalation of polymer length. We describe this escalation theoretically under the conservative assumption of reversible polymerization. Taking into account the separately measured thermophoretic properties of RNA, we extrapolate the results for primordial RNA polymerization inside a temperature gradient in pores or fissures of rocks. With a dilute, nanomolar concentration of monomers the model predicts that a pore length of 5 cm and a temperature difference of 10 K suffice to polymerize 200-mers of RNA in micromolar concentrations. The probability to generate these long RNAs is raised by a factor of >10(600) compared with polymerization in a physical equilibrium. We experimentally validate the theory with the reversible polymerization of DNA blocks in a laser-driven thermal trap. The results confirm that a thermal gradient can significantly enlarge the available sequence space for the emergence of catalytically active polymers.

Toward a unified physical model of nucleosome patterns flanking transcription start sites.

Recent genome-wide maps of nucleosome positions in different eukaryotes revealed patterns around transcription start sites featuring a nucleosome-free region flanked by a periodic modulation of the nucleosome density. For Saccharomyces cerevisiae, the average in vivo pattern was previously shown to be quantitatively described by a "nucleosome gas" model based on the statistical positioning mechanism. However, this simple physical description is challenged by the fact that the pattern differs quantitatively between species and by recent experiments that appear incompatible with statistical positioning, indicating important roles for chromatin remodelers. We undertake a data-driven search for a unified physical model to describe the nucleosome patterns of 12 yeast species and also consider an extension of the model to capture remodeling effects. We are led to a nucleosome gas that takes into account nucleosome breathing, i.e., transient unwrapping of nucleosomal DNA segments. This known biophysical property of nucleosomes rationalizes a "pressure"-induced dependence of the effective nucleosome size that is suggested by the data. By fitting this model to the data, we find an average energy cost for DNA unwrapping consistent with previous biophysical experiments. Although the available data are not sufficient to reconstruct chromatin remodeling mechanisms, a minimal model extension by one mechanism yields an "active nucleosome gas" that can rationalize the behavior of systems with reduced histone-DNA ratio and remodeler knockouts. We therefore establish a basis for a physical description of nucleosome patterns that can serve as a null model for sequence-specific effects at individual genes and in models of transcription regulation.

The phosphorylation flow of the Vibrio harveyi quorum-sensing cascade determines levels of phenotypic heterogeneity in the population.

UNLABELLED: Quorum sensing (QS) is a communication process that enables a bacterial population to coordinate and synchronize specific behaviors. The bioluminescent marine bacterium Vibrio harveyi integrates three autoinducer (AI) signals into one quorum-sensing cascade comprising a phosphorelay involving three hybrid sensor kinases: LuxU; LuxO, an Hfq/small RNA (sRNA) switch; and the transcriptional regulator LuxR. Using a new set of V. harveyi mutants lacking genes for the AI synthases and/or sensors, we assayed the activity of the quorum-sensing cascade at the population and single-cell levels, with a specific focus on signal integration and noise levels. We found that the ratios of kinase activities to phosphatase activities of the three sensors and, hence, the extent of phosphorylation of LuxU/LuxO are important not only for the signaling output but also for the degree of noise in the system. The pools of phosphorylated LuxU/LuxO per cell directly determine the amounts of sRNAs produced and, consequently, the copy number of LuxR, generating heterogeneous quorum-sensing activation at the single-cell level. We conclude that the ability to drive the heterogeneous expression of QS-regulated genes in V. harveyi is an inherent feature of the architecture of the QS cascade. IMPORTANCE: V. harveyi possesses one of the most complex quorum-sensing (QS) cascades known, using three different autoinducers (AIs) to control the induction of, e.g., bioluminescence, virulence factors, and biofilm and exoprotease production. We constructed various V. harveyi mutants to study the impact of each component and subsystem of the QS signaling cascade on QS activation at the population and single-cell levels. We found that the output was homogeneous only in the presence of all AIs. In the absence of any one AI, QS activation varied from cell to cell, resulting in phenotypic heterogeneity. This study elucidates a molecular design principle which enables a tightly integrated signaling cascade to control the expression of diverse phenotypes within a genetically homogeneous population.

Adsorption-Desorption Kinetics of Soft Particles.

Adsorption-desorption processes are ubiquitous in physics, chemistry, and biology. Models usually assume hard particles, but within the realm of soft matter physics the adsorbing particles are compressible. A minimal 1D model reveals that softness fundamentally changes the kinetics: Below the desorption time scale, a logarithmic increase of the particle density replaces the usual Rényi jamming plateau, and the subsequent relaxation to equilibrium can be nonmonotonic and much faster than for hard particles. These effects will impact the kinetics of self-assembly and reaction-diffusion processes.