The bend stiffness of S-DNA

We formulate and solve a two-state model for the elasticity of nicked, double-stranded DNA that borrows features from both the Worm Like Chain and the Bragg--Zimm model. Our model is computationally simple, and gives an excellent fit to recent experimental data through the entire overstretching transition. The fit gives the first value for the bending stiffness of the overstretched state as about 10 nm*kbt, a value quite different from either B-form or single-stranded DNA.Comment: 7 pages, 1 figur

Noise Underlies Switching Behavior of the Bacterial Flagellum

We report the switching behavior of the full bacterial flagellum system that includes the filament and the motor in wild-type Escherichia coli cells. In sorting the motor behavior by the clockwise bias, we find that the distributions of the clockwise (CW) and counterclockwise (CCW) intervals are either exponential or nonexponential with long tails. At low bias, CW intervals are exponentially distributed and CCW intervals exhibit long tails. At intermediate CW bias (0.5) both CW and CCW intervals are mainly exponentially distributed. A simple model suggests that these two distinct switching behaviors are governed by the presence of signaling noise within the chemotaxis network. Low noise yields exponentially distributed intervals, whereas large noise yields nonexponential behavior with long tails. These drastically different motor statistics may play a role in optimizing bacterial behavior for a wide range of environmental conditions.Molecular and Cellular Biolog

Fine-Tuning of Chemotactic Response in E. coli Determined by High-Throughput Capillary Assay

In E. coli, chemotactic behavior exhibits perfect adaptation that is robust to changes in the intracellular concentration of the chemotactic proteins, such as CheR and CheB. However, the robustness of the perfect adaptation does not explicitly imply a robust chemotactic response. Previous studies on the robustness of the chemotactic response relied on swarming assays, which can be confounded by processes besides chemotaxis, such as cellular growth and depletion of nutrients. Here, using a high-throughput capillary assay that eliminates the effects of growth, we experimentally studied how the chemotactic response depends on the relative concentration of the chemotactic proteins. We simultaneously measured both the chemotactic response of E. coli cells to l-aspartate and the concentrations of YFP-CheR and CheB-CFP fusion proteins. We found that the chemotactic response is fine-tuned to a specific ratio of [CheR]/[CheB] with a maximum response comparable to the chemotactic response of wild-type behavior. In contrast to adaptation in chemotaxis, that is robust and exact, capillary assays revealed that the chemotactic response in swimming bacteria is fined-tuned to wild-type level of the [CheR]/[CheB] ratio.Molecular and Cellular Biolog

Uncovering Scaling Laws to Infer Multi-drug Response of Resistant Microbes and Cancer Cells

Drug resistance in bacterial infections and cancers constitutes a major threat to human health. Treatments often include several interacting drugs, but even potent therapies can become ineffective in resistant mutants. Here we simplify the picture of drug resistance by identifying scaling laws that unify the multi-drug responses of drug sensitive and drug resistant cells. Based on these scaling relationships, we are able to infer the two-drug response of resistant mutants in previously unsampled regions of dosage space in clinically relevant microbes such as E. coli, E. faecalis, S. aureus and S. cerevisiae, as well as in human non-small cell lung cancer, melanoma, and breast cancer stem cells. Importantly, we find that scaling relations also apply across evolutionarily close strains. Finally, scaling allows one to rapidly identify new drug combinations and predict potent dosage regimes for targeting resistant mutants without any prior mechanistic knowledge of the specific resistance mechanism.Molecular and Cellular Biolog

A two-state kinetic model for the unfolding of single molecules by mechanical force

We investigate the work dissipated during the irreversible unfolding of single molecules by mechanical force, using the simplest model necessary to represent experimental data. The model consists of two levels (folded and unfolded states) separated by an intermediate barrier. We compute the probability distribution for the dissipated work and give analytical expressions for the average and variance of the distribution. To first order, the amount of dissipated work is directly proportional to the rate of application of force (the loading rate), and to the relaxation time of the molecule. The model yields estimates for parameters that characterize the unfolding kinetics under force in agreement with those obtained in recent experimental results (Liphardt, J., et al. (2002) {\em Science}, {\bf 296} 1832-1835). We obtain a general equation for the minimum number of repeated experiments needed to obtain an equilibrium free energy, to within $k_BT$, from non-equilibrium experiments using the Jarzynski formula. The number of irreversible experiments grows exponentially with the ratio of the average dissipated work, \bar{\Wdis}, to $k_BT$.}Comment: PDF file, 5 page

Theory of High-Force DNA Stretching and Overstretching

Single molecule experiments on single- and double stranded DNA have sparked a renewed interest in the force-extension of polymers. The extensible Freely Jointed Chain (FJC) model is frequently invoked to explain the observed behavior of single-stranded DNA. We demonstrate that this model does not satisfactorily describe recent high-force stretching data. We instead propose a model (the Discrete Persistent Chain, or ``DPC'') that borrows features from both the FJC and the Wormlike Chain, and show that it resembles the data more closely. We find that most of the high-force behavior previously attributed to stretch elasticity is really a feature of the corrected entropic elasticity; the true stretch compliance of single-stranded DNA is several times smaller than that found by previous authors. Next we elaborate our model to allow coexistence of two conformational states of DNA, each with its own stretch and bend elastic constants. Our model is computationally simple, and gives an excellent fit through the entire overstretching transition of nicked, double-stranded DNA. The fit gives the first values for the elastic constants of the stretched state. In particular we find the effective bend stiffness for DNA in this state to be about 10 nm*kbt, a value quite different from either B-form or single-stranded DNAComment: 33 pages, 11 figures. High-quality figures available upon reques

Monitoring lineages of growing and dividing bacteria reveals an inducible memory of mar operon expression

In Gram negative bacteria, the multiple antibiotic resistance or mar operon, is known to control the expression of multi-drug efflux genes that protect bacteria from a wide range of drugs. As many different chemical compounds can induce this operon, identifying the parameters that govern the dynamics of its induction is crucial to better characterize the processes of tolerance and resistance. Most experiments have assumed that the properties of the mar transcriptional network can be inferred from population measurements. However, measurements from an asynchronous population of cells can mask underlying phenotypic variations of single cells. We monitored the activity of the mar promoter in single Escherichia coli cells in linear micro-colonies and established that the response to a steady level of inducer was most heterogeneous within individual colonies for an intermediate value of inducer. Specifically, sub-lineages defined by contiguous daughter-cells exhibited similar promoter activity, whereas activity was greatly variable between different sub-lineages. Specific sub-trees of uniform promoter activity persisted over several generations. Statistical analyses of the lineages suggest that the presence of these sub-trees is the signature of an inducible memory of the promoter state that is transmitted from mother to daughter cells. This single-cell study reveals that the degree of epigenetic inheritance changes as a function of inducer concentration, suggesting that phenotypic inheritance may be an inducible phenotype

Inferring the effective thickness of polyelectrolytes from stretching measurements at various ionic strengths: applications to DNA and RNA

By resorting to the thick-chain model we discuss how the stretching response of a polymer is influenced by the self-avoidance entailed by its finite thickness. The characterization of the force versus extension curve for a thick chain is carried out through extensive stochastic simulations. The computational results are captured by an analytic expression that is used to fit experimental stretching measurements carried out on DNA and single-stranded RNA (poly-U) in various solutions. This strategy allows us to infer the apparent diameter of two biologically-relevant polyelectrolytes, namely DNA and poly-U, for different ionic strengths. Due to the very different degree of flexibility of the two molecules, the results provide insight into how the apparent diameter is influenced by the interplay between the (solution-dependent) Debye screening length and the polymers' ``bare'' thickness. For DNA, the electrostatic contribution to the effective radius, $\Delta$, is found to be about 5 times larger than the Debye screening length, consistently with previous theoretical predictions for highly-charged stiff rods. For the more flexible poly-U chains the electrostatic contribution to $\Delta$ is found to be significantly smaller than the Debye screening length.Comment: iopart, 14 pages, 13 figures, to appear in J. Phys.: Condens. Matte

Conformational spread as a mechanism for cooperativity in the bacterial flagellar switch

The bacterial flagellar switch that controls the direction of flagellar rotation during chemotaxis has a highly cooperative response. This has previously been understood in terms of the classic two-state, concerted model of allosteric regulation. Here, we used high-resolution optical microscopy to observe switching of single motors and uncover the stochastic multistate nature of the switch. Our observations are in detailed quantitative agreement with a recent general model of allosteric cooperativity that exhibits conformational spread—the stochastic growth and shrinkage of domains of adjacent subunits sharing a particular conformational state. We expect that conformational spread will be important in explaining cooperativity in other large signaling complexes

Single molecule experiments in biophysics: exploring the thermal behavior of nonequilibrium small systems

Biomolecules carry out very specialized tasks inside the cell where energies involved are few tens of k_BT, small enough for thermal fluctuations to be relevant in many biomolecular processes. In this paper I discuss a few concepts and present some experimental results that show how the study of fluctuation theorems applied to biomolecules contributes to our understanding of the nonequilibrium thermal behavior of small systems.Comment: Proceedings of the 22nd Statphys Conference 2004 (Bangalore,India). Invited contributio