Joel Stavans

Increased Bending Rigidity of Single DNA Molecules by H-NS, a Temperature and Osmolarity Sensor

Histonelike nucleoid structuring protein (H-NS) is an abundant prokaryotic protein participating in nucleoid structure, gene regulation, and silencing. It plays a key role in cell response to changes in temperature and osmolarity. Force-extension measurements of single, twist-relaxed lambda-DNA-H-NS complexes show that these adopt more extended configurations compared to the naked DNA substrates. Crosslinking indicates that H-NS can decorate DNA molecules at one H-NS dimer per 15-20 bp. These results suggest that H-NS polymerizes along DNA, forming a complex of higher bending rigidity. These effects are not observed above 32 degrees C or at high osmolarity, supporting the hypothesis that a direct H-NS-DNA interaction plays a key role in gene silencing. Thus, we propose that H-NS plays a unique structural role, different from that of HU and IHF, and functions as one of the environmental sensors of the cell.

Precise temporal modulation in the response of the SOS DNA repair network in individual bacteria.

The SOS genetic network is responsible for the repair/bypass of DNA damage in bacterial cells. While the initial stages of the response have been well characterized, less is known about the dynamics of the response after induction and its shutoff. To address this, we followed the response of the SOS network in living individual Escherichia coli cells. The promoter activity (PA) of SOS genes was monitored using fluorescent protein-promoter fusions, with high temporal resolution, after ultraviolet irradiation activation. We find a temporal pattern of discrete activity peaks masked in studies of cell populations. The number of peaks increases, while their amplitude reaches saturation, as the damage level is increased. Peak timing is highly precise from cell to cell and is independent of the stage in the cell cycle at the time of damage. Evidence is presented for the involvement of the umuDC operon in maintaining the pattern of PA and its temporal precision, providing further evidence for the role UmuD cleavage plays in effecting a timed pause during the SOS response, as previously proposed. The modulations in PA we observe share many features in common with the oscillatory behavior recently observed in a mammalian DNA damage response. Our results, which reveal a hitherto unknown modulation of the SOS response, underscore the importance of carrying out dynamic measurements at the level of individual living cells in order to unravel how a natural genetic network operates at the systems level.

The evolution of cellular structures

A large class of evolving nonequilibrium systems, known collectively as cellular structures, are composed of nearly-uniform domains of polygonal-like or polyhedral-like shape (in two- or three-dimensional systems respectively) separated by thin boundaries endowed with line or surface energy. Work done mainly during the last decade has shown that the evolution of mature structures is characterized by universal or system-independent statistical distributions which possess scaling properties. The author presents an introduction to cellular structures, discusses the fundamental role played by geometry in the evolution of these systems and surveys the recent experimental and theoretical developments in the field.

Compaction of single DNA molecules induced by binding of integration host factor (IHF)

We studied the interaction between the integration host factor (IHF), a major nucleoid-associated protein in bacteria, and single DNA molecules. Force–extension measurements of λ DNA and an analysis of the Brownian motion of small beads tethered to a surface by single short DNA molecules, in equilibrium with an IHF solution, indicate that: (i) the DNA–IHF complex retains a random, although more compact, coiled configuration for zero or small values of the tension, (ii) IHF induces DNA compaction by binding to multiple DNA sites with low specificity, and (iii) with increasing tension on the DNA, the elastic properties of bare DNA are recovered. This behavior is consistent with the predictions of a statistical mechanical model describing how proteins bending DNA are driven off by an applied tension on the DNA molecule. Estimates of the amount of bound IHF in DNA–IHF complexes obtained from the model agree very well with independent measurements of this quantity obtained from the analysis of DNA–IHF crosslinking. Our findings support the long-held view that IHF and other histone-like proteins play an important role in shaping the long-scale structure of the bacterial nucleoid.

Pearling Instabilities of Membrane Tubes with Anchored Polymers

We have studied the pearling instability induced on hollow tubular lipid vesicles by hydrophilic polymers with hydrophobic side groups along the backbone. The results show that the polymer concentration is coupled to local membrane curvature. The relaxation of a pearled tube is characterized by two different well-separated time scales, indicating two physical mechanisms. We present a model, which explains the observed phenomena and predicts polymer segregation according to local membrane curvature at late stages.

DNA–protein interactions and bacterial chromosome architecture

Bacteria, like eukaryotic organisms, must compact the DNA molecule comprising their genome and form a functional chromosome. Yet, bacteria do it differently. A number of factors contribute to genome compaction and organization in bacteria, including entropic effects, supercoiling and DNA-protein interactions. A gamut of new experimental techniques have allowed new advances in the investigation of these factors, and spurred much interest in the dynamic response of the chromosome to environmental cues, segregation, and architecture, during both exponential and stationary phases. We review these recent developments with emphasis on the multifaceted roles that DNA-protein interactions play.

Objective-type dark-field illumination for scattering from microbeads

We introduce a method for detecting and tracking small particles in a solution near a surface. The method is based on blocking the backreflected illumination beam in an objective-type total internal reflection microscope, leaving unhindered the light scattered by the particles and resulting in dark-field illumination. Using this method, we tracked the motion of 60-nm polystyrene beads with a signal-to-noise ratio of 6 and detected 20-nm gold particles with a signal-to-noise ratio of 5. We illustrate the methods use by following the Brownian motion of small beads attached by short DNA tethers to a substrate.

Self-Diffusion in Granular Flows

The mixing of identical grains in a granular flow was experimentally studied and found to be a self-diffusive process. The flow, produced by vertically vibrating a horizontal layer of grains, is two-dimensional allowing us to study self-diffusion in the perpendicular direction to the flow plane. The self-diffusion coefficient depends only on the flow velocity and not on the independent values of frequency and amplitude of vibration. Its growth with the flow velocity is consistent with a linear dependence. Our findings support the picture of the velocity field as consisting of convective and fluctuating parts and agree qualitatively with recent hydrodynamical models of granular flows.

Mobility of a Sphere in Vibrated Granular Media

We report the results of the first measurements of the mobility/friction coefficients of a sphere dragged horizontally through a vertically vibrated granular medium. The friction coefficient depends on the state of excitation of the medium and drops rapidly when the acceleration of the vibrated cell exceeds that of gravity. We propose a simple model involving fluidization/solidification events during a period of vibration of the cell. We argue that the observed mobility is proportional to the fraction of the time spent in the fluid state and discuss the origin of the observed dependence on the various parameters.

Coiling of Cylindrical Membrane Stacks with Anchored Polymers

(Received 22 April 1999) We study experimentally a coiling instability of cylindrical multilamellar stacks of phospholipid membranes, induced by polymers with hydrophobic anchors grafted along their hydrophilic backbone. We interpret our experimental results in terms of a model in which local membrane curvature and polymer concentration are coupled. The model predicts the occurrence of maximally tight coils above a threshold polymer concentration. Indeed, only maximally tight coils are observed experimentally. Our system is unique in that coils form in the absence of twist and adhesion. The coil motif is ubiquitous in a wide range of natural contexts. One-dimensional filaments of mutant bacteria [1], supercoiled DNA molecules [2], and tendrils of climbing plants [3] all exhibit a writhing instability as a result of forcing or interaction with an external agent. Such systems are dominated by elastic properties, and the appearance of coils is a result of the relief of twist. In this paper we show that coiling can also be effected in cylindrical multilamellar tubes of phospholipid bilayers, by anchoring hydrophilic polymers with hydrophobic side groups grafted along the backbone. This system is unique in that, in contrast with the above examples, fluid membranes cannot support any twist. Yet coils are