Stanislas Leibler

A synthetic oscillatory network of transcriptional regulators

Networks of interacting biomolecules carry out many essential functions in living cells, but the ‘design principles’ underlying the functioning of such intracellular networks remain poorly understood, despite intensive efforts including quantitative analysis of relatively simple systems. Here we present a complementary approach to this problem: the design and construction of a synthetic network to implement a particular function. We used three transcriptional repressor systems that are not part of any natural biological clock to build an oscillating network, termed the repressilator, in Escherichia coli. The network periodically induces the synthesis of green fluorescent protein as a readout of its state in individual cells. The resulting oscillations, with typical periods of hours, are slower than the cell-division cycle, so the state of the oscillator has to be transmitted from generation to generation. This artificial clock displays noisy behaviour, possibly because of stochastic fluctuations of its components. Such ‘rational network design’ may lead both to the engineering of new cellular behaviours and to an improved understanding of naturally occurring networks.

Mechanisms of noise-resistance in genetic oscillators

A wide range of organisms use circadian clocks to keep internal sense of daily time and regulate their behavior accordingly. Most of these clocks use intracellular genetic networks based on positive and negative regulatory elements. The integration of these “circuits” at the cellular level imposes strong constraints on their functioning and design. Here, we study a recently proposed model [Barkai, N. & Leibler, S. (2000) Nature (London), 403, 267–268] that incorporates just the essential elements found experimentally. We show that this type of oscillator is driven mainly by two elements: the concentration of a repressor protein and the dynamics of an activator protein forming an inactive complex with the repressor. Thus, the clock does not need to rely on mRNA dynamics to oscillate, which makes it especially resistant to fluctuations. Oscillations can be present even when the time average of the number of mRNA molecules goes below one. Under some conditions, this oscillator is not only resistant to but, paradoxically, also enhanced by the intrinsic biochemical noise.

Circadian clocks limited by noise.

Circadian rhythms, which provide internal daily periodicity, are used by a wide range of organisms to anticipate daily changes in the environment. It seems that these organisms generate circadian periodicity by similar biochemical networks within a single cell. A model based on the common features of these biochemical networks shows that a circadian network can oscillate reliably in the presence of stochastic biochemical noise and when cellular conditions are altered. We propose that the ability to resist such perturbations imposes strict constraints on the oscillation mechanisms underlying circadian periodicity in vivo.

Self-organization of microtubules and motors

Cellular structures are established and maintained through a dynamic interplay between assembly and regulatory processes. Self-organization of molecular components provides a variety of possible spatial structures: the regulatory machinery chooses the most appropriate to express a given cellular function. Here we study the extent and the characteristics of self-organization using microtubules and molecular motors as a model system. These components are known to participate in the formation of many cellular structures, such as the dynamic asters found in mitotic and meiotic spindles. Purified motors and microtubules have previously been observed to form asters in vitro. We have reproduced this result with a simple system consisting solely of multi-headed constructs of the motor protein kinesin and stabilized microtubules. We show that dynamic asters can also be obtained from a homogeneous solution of tubulin and motors. By varying the relative concentrations of the components, we obtain a variety of self-organized structures. Further, by studying this process in a constrained geometry of micro-fabricated glass chambers, we demonstrate that the same final structure can be reached through different assembly ‘pathways’.

Dynamic persistence of antibiotic-stressed mycobacteria.

All About Noise How individual cells behave within a larger “average” population can be surprising. Wakamoto et al. (p. 91) developed a method for investigating the consequences of phenotypic variability in single mycobacterial cells exposed to the pro-drug, isoniazid. Isoniazid needs to be activated by bacterial catalase. In the isoniazid–mycobacterium system, random fluctuations in catalase activity were important for cell survival. Because catalase is essential, it cannot be ablated; however, catalase activity pulsed randomly in the mycobacteria. Thus, a subpopulation of individual cells manage to avoid being killed by the activated antibiotic. Bacterial cells that divide before a pulse of catalase expression survive the application of the antibiotic isoniazid. Exposure of an isogenic bacterial population to a cidal antibiotic typically fails to eliminate a small fraction of refractory cells. Historically, fractional killing has been attributed to infrequently dividing or nondividing “persisters.” Using microfluidic cultures and time-lapse microscopy, we found that Mycobacterium smegmatis persists by dividing in the presence of the drug isoniazid (INH). Although persistence in these studies was characterized by stable numbers of cells, this apparent stability was actually a dynamic state of balanced division and death. Single cells expressed catalase-peroxidase (KatG), which activates INH, in stochastic pulses that were negatively correlated with cell survival. These behaviors may reflect epigenetic effects, because KatG pulsing and death were correlated between sibling cells. Selection of lineages characterized by infrequent KatG pulsing could allow nonresponsive adaptation during prolonged drug exposure.

Simpson's paradox in a synthetic microbial system.

The maintenance of “public” or “common good” producers is a major question in the evolution of cooperation. Because nonproducers benefit from the shared resource without bearing its cost of production, they may proliferate faster than producers. We established a synthetic microbial system consisting of two Escherichia coli strains of common-good producers and nonproducers. Depending on the population structure, which was varied by forming groups with different initial compositions, an apparently paradoxical situation could be attained in which nonproducers grew faster within each group, yet producers increased overall. We show that a simple way to generate the variance required for this effect is through stochastic fluctuations via population bottlenecks. The synthetic approach described here thus provides a way to study generic mechanisms of natural selection.

Response regulator output in bacterial chemotaxis

Chemotaxis responses in Escherichia coli are mediated by the phosphorylated response‐regulator protein P‐CheY. Biochemical and genetic studies have established the mechanisms by which the various components of the chemotaxis system, the membrane receptors and Che proteins function to modulate levels of CheY phosphorylation. Detailed models have been formulated to explain chemotaxis sensing in quantitative terms; however, the models cannot be adequately tested without knowledge of the quantitative relationship between P‐CheY and bacterial swimming behavior. A computerized image analysis system was developed to collect extensive statistics on freeswimming and individual tethered cells. P‐CheY levels were systematically varied by controlled expression of CheY in an E.coli strain lacking the CheY phosphatase, CheZ, and the receptor demethylating enzyme CheB. Tumbling frequency was found to vary with P‐CheY concentration in a weakly sigmoidal fashion (apparent Hill coefficient ∼2.5). This indicates that the high sensitivity of the chemotaxis system is not derived from highly cooperative interactions between P‐CheY and the flagellar motor, but rather depends on nonlinear effects within the chemotaxis signal transduction network. The complex relationship between single flagella rotation and free‐swimming behavior was examined; our results indicate that there is an additional level of information processing associated with interactions between the individual flagella. An allosteric model of the motor switching process is proposed which gives a good fit to the observed switching induced by P‐CheY. Thus the level of intracellular P‐CheY can be estimated from behavior determinations: ∼30% of the intracellular pool of CheY appears to be phosphorylated in fully adapted wild‐type cells.

A MAGNETIC MANIPULATOR FOR STUDYING LOCAL RHEOLOGY AND MICROMECHANICAL PROPERTIES OF BIOLOGICAL SYSTEMS

A magnetic micromanipulator capable of generating two‐dimensional translational and rotational motions on a microscope stage is described. With 3 μm‐diam paramagnetic beads, forces in the piconewton range and torques on the order of 10−14 N m are obtained and can be modulated in time at moderate frequencies (<5 Hz). Typical magnetic fields between 0.1 and 0.2 T, and gradients between 10 and 20 T m−1 are created by four independent feedback‐controlled electromagnets. Video microscopy and computerized image analysis are used to locate the beads on each image with a resolution of 0.1 pixel (20 nm). The device is primarily designed to study, at a microscopic scale, the local mechanical properties of biological polymers such as actin in solution, and of cell cytoplasm. Possible applications include the in situ manipulation of intracellular organelles.

Photoactivation turns green fluorescent protein red

In the few years since its gene was first cloned, the Aequorea victoria green fluorescent protein (GFP) has become a powerful tool in cell biology, functioning as a marker for gene expression, protein localization and protein dynamics in living cells. GFP variants with improved fluorescence intensity and altered spectral characteristics have been identified, but additional GFP variants are still desirable for multiple labeling experiments, protein interaction studies and improved visibility in some organisms. In particular, long-wavelength (red) fluorescence has remained elusive. Here we describe a red-emitting, green-absorbing fluorescent state of GFP that is generated by photoactivation with blue light. GFP can be switched to its red-emitting state easily with a laser or fluorescence microscope lamp under conditions of low oxygen concentration. This previously unnoticed ability enables regional, non-invasive marking of proteins in vivo. In particular, we report here the use of GFP photoactivation to make the first direct measurements of protein diffusion in the cytoplasm of living bacteria.

Environmental stresses can alleviate the average deleterious effect of mutations

Background Fundamental questions in evolutionary genetics, including the possible advantage of sexual reproduction, depend critically on the effects of deleterious mutations on fitness. Limited existing experimental evidence suggests that, on average, such effects tend to be aggravated under environmental stresses, consistent with the perception that stress diminishes the organisms ability to tolerate deleterious mutations. Here, we ask whether there are also stresses with the opposite influence, under which the organism becomes more tolerant to mutations. Results We developed a technique, based on bioluminescence, which allows accurate automated measurements of bacterial growth rates at very low cell densities. Using this system, we measured growth rates of Escherichia coli mutants under a diverse set of environmental stresses. In contrast to the perception that stress always reduces the organisms ability to tolerate mutations, our measurements identified stresses that do the opposite – that is, despite decreasing wild-type growth, they alleviate, on average, the effect of deleterious mutations. Conclusions Our results show a qualitative difference between various environmental stresses ranging from alleviation to aggravation of the average effect of mutations. We further show how the existence of stresses that are biased towards alleviation of the effects of mutations may imply the existence of average epistatic interactions between mutations. The results thus offer a connection between the two main factors controlling the effects of deleterious mutations: environmental conditions and epistatic interactions.