Alon Zaslaver

A comprehensive library of fluorescent transcriptional reporters for Escherichia coli

E. coli is widely used for systems biology research; there exists a need, however, for tools that can be used to accurately and comprehensively measure expression dynamics in individual living cells. To address this we present a library of transcriptional fusions of gfp to each of about 2,000 different promoters in E. coli K12, covering the great majority of the promoters in the organism. Each promoter fusion is expressed from a low-copy plasmid. We demonstrate that this library can be used to obtain highly accurate dynamic measurements of promoter activity on a genomic scale, in a glucose-lactose diauxic shift experiment. The library allowed detection of about 80 previously uncharacterized transcription units in E. coli, including putative internal promoters within previously known operons, such as the lac operon. This library can serve as a tool for accurate, high-resolution analysis of transcription networks in living E. coli cells.

Plasticity of the cis-regulatory input function of a gene.

The transcription rate of a gene is often controlled by several regulators that bind specific sites in the genes cis-regulatory region. The combined effect of these regulators is described by a cis-regulatory input function. What determines the form of an input function, and how variable is it with respect to mutations? To address this, we employ the well-characterized lac operon of Escherichia coli, which has an elaborate input function, intermediate between Boolean AND-gate and OR-gate logic. We mapped in detail the input function of 12 variants of the lac promoter, each with different point mutations in the regulator binding sites, by means of accurate expression measurements from living cells. We find that even a few mutations can significantly change the input function, resulting in functions that resemble Pure AND gates, OR gates, or single-input switches. Other types of gates were not found. The variant input functions can be described in a unified manner by a mathematical model. The model also lets us predict which functions cannot be reached by point mutations. The input function that we studied thus appears to be plastic, in the sense that many of the mutations do not ruin the regulation completely but rather result in new ways to integrate the inputs.

Cost of Unneeded Proteins in E. coli Is Reduced after Several Generations in Exponential Growth

When E. coli cells express unneeded protein, they grow more slowly. Such penalty to fitness associated with making proteins is called protein cost. Protein cost is an important component in the cost-benefit tradeoffs that underlie the evolution of protein circuits, but its origins are still poorly understood. Here, we ask how the protein cost varies during the exponential growth phase of E. coli. We find that cells growing exponentially following an upshift from overnight culture show a large cost when producing unneeded proteins. However, after several generations, while still in exponential growth, the cells enter a phase where cost is much reduced despite vigorous unneeded protein production. We find that this reduced-cost phase depends on the ppGpp system, which adjusts the amount of ribosomes in the cell and does not occur after a downshift from rich to poor medium. These findings suggest that protein cost is a transient phenomenon that happens upon an upshift in conditions and that cost is reduced when ribosomes and other cellular systems have increased to their appropriate steady-state level in the new condition.

Diverse Two-Dimensional Input Functions Control Bacterial Sugar Genes

Cells respond to signals by regulating gene expression. The relation between the level of input signals and the transcription rate of the gene is called the genes input function. Because most genes are regulated by more than one signal, the input functions are usually multidimensional. To understand cellular responses, it is essential to know the shapes of these functions. Here, we map the two-dimensional input functions of 19 sugar-utilization genes at high resolution in living E. coli cells. We find diverse, intricately shaped input functions, despite the similarity in the regulatory circuitry of these genes. Surprisingly, some of the input functions are nonmonotonic, peaking at intermediate signal levels. Furthermore, most of the input functions show separation of variables, in the sense that they can be described as the product of simple functions that depend on a single input. This first broad survey of two-dimensional input functions can be extended to map the logic of gene regulation in other systems.

A modular library of small molecule signals regulates social behaviors in Caenorhabditis elegans.

Comparative metabolomics reveals a modular library of small molecule signals that function as aggregation pheromones in the nematode C. elegans.

Invariant Distribution of Promoter Activities in Escherichia coli

Cells need to allocate their limited resources to express a wide range of genes. To understand how Escherichia coli partitions its transcriptional resources between its different promoters, we employ a robotic assay using a comprehensive reporter strain library for E. coli to measure promoter activity on a genomic scale at high-temporal resolution and accuracy. This allows continuous tracking of promoter activity as cells change their growth rate from exponential to stationary phase in different media. We find a heavy-tailed distribution of promoter activities, with promoter activities spanning several orders of magnitude. While the shape of the distribution is almost completely independent of the growth conditions, the identity of the promoters expressed at different levels does depend on them. Translation machinery genes, however, keep the same relative expression levels in the distribution across conditions, and their fractional promoter activity tracks growth rate tightly. We present a simple optimization model for resource allocation which suggests that the observed invariant distributions might maximize growth rate. These invariant features of the distribution of promoter activities may suggest design constraints that shape the allocation of transcriptional resources.

Metazoan Operons Accelerate Recovery from Growth-Arrested States

Existing theories explain why operons are advantageous in prokaryotes, but their occurrence in metazoans is an enigma. Nematode operon genes, typically consisting of growth genes, are significantly upregulated during recovery from growth-arrested states. This expression pattern is anticorrelated to nonoperon genes, consistent with a competition for transcriptional resources. We find that transcriptional resources are initially limiting during recovery and that recovering animals are highly sensitive to any additional decrease in transcriptional resources. We provide evidence that operons become advantageous because, by clustering growth genes into operons, fewer promoters compete for the limited transcriptional machinery, effectively increasing the concentration of transcriptional resources and accelerating recovery. Mathematical modeling reveals how a moderate increase in transcriptional resources can substantially enhance transcription rate and recovery. This design principle occurs in different nematodes and the chordate C. intestinalis. As transition from arrest to rapid growth is shared by many metazoans, operons could have evolved to facilitate these processes.

Hierarchical sparse coding in the sensory system of Caenorhabditis elegans

Significance We investigated how a numerically and spatially compact nematode nervous system encodes information about the world. A library of transgenic worms expressing a genetically encoded calcium indicator in each type of sensory neuron was constructed and used to assay neural activity in response to various chemical stimuli to compile a functional map of a sensory system. We find that the sensory system uses hierarchical sparse coding, a strategy that mitigates the limited size and the shallow structure of the neural network. Also, this is a timely study that significantly adds to the communal effort and enthusiasm in obtaining functional maps of the connectome. Animals with compact sensory systems face an encoding problem where a small number of sensory neurons are required to encode information about its surrounding complex environment. Using Caenorhabditis elegans worms as a model, we ask how chemical stimuli are encoded by a small and highly connected sensory system. We first generated a comprehensive library of transgenic worms where each animal expresses a genetically encoded calcium indicator in individual sensory neurons. This library includes the vast majority of the sensory system in C. elegans. Imaging from individual sensory neurons while subjecting the worms to various stimuli allowed us to compile a comprehensive functional map of the sensory system at single neuron resolution. The functional map reveals that despite the dense wiring, chemosensory neurons represent the environment using sparse codes. Moreover, although anatomically closely connected, chemo- and mechano-sensory neurons are functionally segregated. In addition, the code is hierarchical, where few neurons participate in encoding multiple cues, whereas other sensory neurons are stimulus specific. This encoding strategy may have evolved to mitigate the constraints of a compact sensory system.

Serotonin promotes exploitation in complex environments by accelerating decision-making.

BackgroundFast responses can provide a competitive advantage when resources are inhomogeneously distributed. The nematode Caenorhabditis elegans was shown to modulate locomotion on a lawn of bacterial food in serotonin (5-HT)-dependent manners. However, potential roles for serotonergic signaling in responding to food discovery are poorly understood.ResultsWe found that 5-HT signaling in C. elegans facilitates efficient exploitation in complex environments by mediating a rapid response upon encountering food. Genetic or cellular manipulations leading to deficient serotonergic signaling resulted in gradual responses and defective exploitation of a patchy foraging landscape. Physiological imaging revealed that the NSM serotonergic neurons responded acutely upon encounter with newly discovered food and were key to rapid responses. In contrast, the onset of responses of ADF serotonergic neurons preceded the physical encounter with the food. The serotonin-gated chloride channel MOD-1 and the ortholog of mammalian 5-HT1 metabotropic serotonin receptors SER-4 acted in synergy to accelerate decision-making. The relevance of responding rapidly was demonstrated in patchy environments, where the absence of 5-HT signaling was detrimental to exploitation.ConclusionsOur results implicate 5-HT in a novel form of decision-making, demonstrate its fitness consequences, suggest that NSM and ADF act in concert to modulate locomotion in complex environments, and identify the synergistic action of a channel and a metabotropic receptor in accelerating C. elegans decision-making.

Salient experiences are represented by unique transcriptional signatures in the mouse brain

It is well established that inducible transcription is essential for the consolidation of salient experiences into long-term memory. However, whether inducible transcription relays information about the identity and affective attributes of the experience being encoded, has not been explored. To this end, we analyzed transcription induced by a variety of rewarding and aversive experiences, across multiple brain regions. Our results describe the existence of robust transcriptional signatures uniquely representing distinct experiences, enabling near-perfect decoding of recent experiences. Furthermore, experiences with shared attributes display commonalities in their transcriptional signatures, exemplified in the representation of valence, habituation and reinforcement. This study introduces the concept of a neural transcriptional code, which represents the encoding of experiences in the mouse brain. This code is comprised of distinct transcriptional signatures that correlate to attributes of the experiences that are being committed to long-term memory.