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.

Adaptation dynamics between copy-number and point mutations.

Together, copy-number and point mutations form the basis for most evolutionary novelty, through the process of gene duplication and divergence. While a plethora of genomic data reveals the long-term fate of diverging coding sequences and their cis-regulatory elements, little is known about the early dynamics around the duplication event itself. In microorganisms, selection for increased gene expression often drives the expansion of gene copy-number mutations, which serves as a crude adaptation, prior to divergence through refining point mutations. Using a simple synthetic genetic reporter system that can distinguish between copy-number and point mutations, we study their early and transient adaptive dynamics in real time in Escherichia coli. We find two qualitatively different routes of adaptation, depending on the level of functional improvement needed. In conditions of high gene expression demand, the two mutation types occur as a combination. However, under low gene expression demand, copy-number and point mutations are mutually exclusive; here, owing to their higher frequency, adaptation is dominated by copy-number mutations, in a process we term amplification hindrance. Ultimately, due to high reversal rates and pleiotropic cost, copy-number mutations may not only serve as a crude and transient adaptation, but also constrain sequence divergence over evolutionary time scales.

Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14.

A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the host's immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on mouse dendritic cells (DCs) as a binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of the pathogenic strain CFT073 to CD14 reduced DC migration by overactivation of integrins and blunted expression of co-stimulatory molecules by overactivating the NFAT (nuclear factor of activated T-cells) pathway, both rate-limiting factors of T cell activation. This response was binary at the single-cell level, but averaged in larger populations exposed to both piliated and non-piliated pathogens, presumably via the exchange of immunomodulatory cytokines. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohn's disease.

Predicting bacterial promoter function and evolution from random sequences.

Predicting function from sequence is a central problem of biology. Currently, this is possible only locally in a narrow mutational neighborhood around a wildtype sequence rather than globally from any sequence. Using random mutant libraries, we developed a biophysical model that accounts for multiple features of σ70 binding bacterial promoters to predict constitutive gene expression levels from any sequence. We experimentally and theoretically estimated that 10-20% of random sequences lead to expression and ~80% of non-expressing sequences are one mutation away from a functional promoter. The potential for generating expression from random sequences is so pervasive that selection acts against σ70-RNA polymerase binding sites even within inter-genic, promoter-containing regions. This pervasiveness of σ70-binding sites implies that emergence of promoters is not the limiting step in gene regulatory evolution. Ultimately, the inclusion of novel features of promoter function into a mechanistic model enabled not only more accurate predictions of gene expression levels, but also identified that promoters evolve more rapidly than previously thought.

Local genetic context shapes the function of a gene regulatory network.

Gene expression levels are influenced by multiple coexisting molecular mechanisms. Some of these interactions such as those of transcription factors and promoters have been studied extensively. However, predicting phenotypes of gene regulatory networks (GRNs) remains a major challenge. Here, we use a well-defined synthetic GRN to study in Escherichia coli how network phenotypes depend on local genetic context, i.e. the genetic neighborhood of a transcription factor and its relative position. We show that one GRN with fixed topology can display not only quantitatively but also qualitatively different phenotypes, depending solely on the local genetic context of its components. Transcriptional read-through is the main molecular mechanism that places one transcriptional unit (TU) within two separate regulons without the need for complex regulatory sequences. We propose that relative order of individual TUs, with its potential for combinatorial complexity, plays an important role in shaping phenotypes of GRNs.

Evolutionary potential of transcription factors for gene regulatory rewiring.

Gene regulatory networks evolve through rewiring of individual components-that is, through changes in regulatory connections. However, the mechanistic basis of regulatory rewiring is poorly understood. Using a canonical gene regulatory system, we quantify the properties of transcription factors that determine the evolutionary potential for rewiring of regulatory connections: robustness, tunability and evolvability. In vivo repression measurements of two repressors at mutated operator sites reveal their contrasting evolutionary potential: while robustness and evolvability were positively correlated, both were in trade-off with tunability. Epistatic interactions between adjacent operators alleviated this trade-off. A thermodynamic model explains how the differences in robustness, tunability and evolvability arise from biophysical characteristics of repressor-DNA binding. The model also uncovers that the energy matrix, which describes how mutations affect repressor-DNA binding, encodes crucial information about the evolutionary potential of a repressor. The biophysical determinants of evolutionary potential for regulatory rewiring constitute a mechanistic framework for understanding network evolution.

Statistical mechanics for metabolic networks during steady state growth.

Which properties of metabolic networks can be derived solely from stoichiometry? Predictive results have been obtained by flux balance analysis (FBA), by postulating that cells set metabolic fluxes to maximize growth rate. Here we consider a generalization of FBA to single-cell level using maximum entropy modeling, which we extend and test experimentally. Specifically, we define for Escherichia coli metabolism a flux distribution that yields the experimental growth rate: the model, containing FBA as a limit, provides a better match to measured fluxes and it makes a wide range of predictions: on flux variability, regulation, and correlations; on the relative importance of stoichiometry vs. optimization; on scaling relations for growth rate distributions. We validate the latter here with single-cell data at different sub-inhibitory antibiotic concentrations. The model quantifies growth optimization as emerging from the interplay of competitive dynamics in the population and regulation of metabolism at the level of single cells.

Leaky resistance and the conditions for the existence of lytic bacteriophage.

In experimental cultures, when bacteria are mixed with lytic (virulent) bacteriophage, bacterial cells resistant to the phage commonly emerge and become the dominant population of bacteria. Following the ascent of resistant mutants, the densities of bacteria in these simple communities become limited by resources rather than the phage. Despite the evolution of resistant hosts, upon which the phage cannot replicate, the lytic phage population is most commonly maintained in an apparently stable state with the resistant bacteria. Several mechanisms have been put forward to account for this result. Here we report the results of population dynamic/evolution experiments with a virulent mutant of phage Lambda, λVIR, and Escherichia coli in serial transfer cultures. We show that, following the ascent of λVIR-resistant bacteria, λVIR is maintained in the majority of cases in maltose-limited minimal media and in all cases in nutrient-rich broth. Using mathematical models and experiments, we show that the dominant mechanism responsible for maintenance of λVIR in these resource-limited populations dominated by resistant E. coli is a high rate of either phenotypic or genetic transition from resistance to susceptibility-a hitherto undemonstrated mechanism we term "leaky resistance." We discuss the implications of leaky resistance to our understanding of the conditions for the maintenance of phage in populations of bacteria-their "existence conditions."

Lack of cations in flow cytometry buffers affect fluorescence signals by reducing membrane stability and viability of Escherichia coli strains.

Buffers are essential for diluting bacterial cultures for flow cytometry analysis in order to study bacterial physiology and gene expression parameters based on fluorescence signals. Using a variety of constitutively expressed fluorescent proteins in Escherichia coli K-12 strain MG1655, we found strong artifactual changes in fluorescence levels after dilution into the commonly used flow cytometry buffer phosphate-buffered saline (PBS) and two other buffer solutions, Tris-HCl and M9 salts. These changes appeared very rapidly after dilution, and were linked to increased membrane permeability and loss in cell viability. We observed buffer-related effects in several different E. coli strains, K-12, C and W, but not E. coli B, which can be partially explained by differences in lipopolysaccharide (LPS) and outer membrane composition. Supplementing the buffers with divalent cations responsible for outer membrane stability, Mg2+ and Ca2+, preserved fluorescence signals, membrane integrity and viability of E. coli. Thus, stabilizing the bacterial outer membrane is essential for precise and unbiased measurements of fluorescence parameters using flow cytometry.

Phage-host population dynamics promotes prophage acquisition in bacteria with innate immunity.

Temperate bacteriophages integrate in bacterial genomes as prophages and represent an important source of genetic variation for bacterial evolution, frequently transmitting fitness-augmenting genes such as toxins responsible for virulence of major pathogens. However, only a fraction of bacteriophage infections are lysogenic and lead to prophage acquisition, whereas the majority are lytic and kill the infected bacteria. Unless able to discriminate lytic from lysogenic infections, mechanisms of immunity to bacteriophages are expected to act as a double-edged sword and increase the odds of survival at the cost of depriving bacteria of potentially beneficial prophages. We show that although restriction-modification systems as mechanisms of innate immunity prevent both lytic and lysogenic infections indiscriminately in individual bacteria, they increase the number of prophage-acquiring individuals at the population level. We find that this counterintuitive result is a consequence of phage-host population dynamics, in which restriction-modification systems delay infection onset until bacteria reach densities at which the probability of lysogeny increases. These results underscore the importance of population-level dynamics as a key factor modulating costs and benefits of immunity to temperate bacteriophages.

Effects of mutations in phage restriction sites during escape from restriction-modification.

Restriction-modification systems are widespread genetic elements that protect bacteria from bacteriophage infections by recognizing and cleaving heterologous DNA at short, well-defined sequences called restriction sites. Bioinformatic evidence shows that restriction sites are significantly underrepresented in bacteriophage genomes, presumably because bacteriophages with fewer restriction sites are more likely to escape cleavage by restriction-modification systems. However, how mutations in restriction sites affect the likelihood of bacteriophage escape is unknown. Using the bacteriophage λ and the restriction-modification system EcoRI, we show that while mutation effects at different restriction sites are unequal, they are independent. As a result, the probability of bacteriophage escape increases with each mutated restriction site. Our results experimentally support the role of restriction site avoidance as a response to selection imposed by restriction-modification systems and offer an insight into the events underlying the process of bacteriophage escape.

Shaping bacterial population behavior through computer-interfaced control of individual cells.

Bacteria in groups vary individually, and interact with other bacteria and the environment to produce population-level patterns of gene expression. Investigating such behavior in detail requires measuring and controlling populations at the single-cell level alongside precisely specified interactions and environmental characteristics. Here we present an automated, programmable platform that combines image-based gene expression and growth measurements with on-line optogenetic expression control for hundreds of individual Escherichia coli cells over days, in a dynamically adjustable environment. This integrated platform broadly enables experiments that bridge individual and population behaviors. We demonstrate: (i) population structuring by independent closed-loop control of gene expression in many individual cells, (ii) cell-cell variation control during antibiotic perturbation, (iii) hybrid bio-digital circuits in single cells, and freely specifiable digital communication between individual bacteria. These examples showcase the potential for real-time integration of theoretical models with measurement and control of many individual cells to investigate and engineer microbial population behavior.

Regulatory network structure determines patterns of intermolecular epistasis.

Most phenotypes are determined by molecular systems composed of specifically interacting molecules. However, unlike for individual components, little is known about the distributions of mutational effects of molecular systems as a whole. We ask how the distribution of mutational effects of a transcriptional regulatory system differs from the distributions of its components, by first independently, and then simultaneously, mutating a transcription factor and the associated promoter it represses. We find that the system distribution exhibits increased phenotypic variation compared to individual component distributions - an effect arising from intermolecular epistasis between the transcription factor and its DNA-binding site. In large part, this epistasis can be qualitatively attributed to the structure of the transcriptional regulatory system and could therefore be a common feature in prokaryotes. Counter-intuitively, intermolecular epistasis can alleviate the constraints of individual components, thereby increasing phenotypic variation that selection could act on and facilitating adaptive evolution.

An Effective Feedback Loop between Cell-Cell Contact Duration and Morphogen Signaling Determines Cell Fate.

Cell-cell contact formation constitutes an essential step in evolution, leading to the differentiation of specialized cell types. However, remarkably little is known about whether and how the interplay between contact formation and fate specification affects development. Here, we identify a positive feedback loop between cell-cell contact duration, morphogen signaling, and mesendoderm cell-fate specification during zebrafish gastrulation. We show that long-lasting cell-cell contacts enhance the competence of prechordal plate (ppl) progenitor cells to respond to Nodal signaling, required for ppl cell-fate specification. We further show that Nodal signaling promotes ppl cell-cell contact duration, generating a positive feedback loop between ppl cell-cell contact duration and cell-fate specification. Finally, by combining mathematical modeling and experimentation, we show that this feedback determines whether anterior axial mesendoderm cells become ppl or, instead, turn into endoderm. Thus, the interdependent activities of cell-cell signaling and contact formation control fate diversification within the developing embryo.

Complex chromosomal neighborhood effects determine the adaptive potential of a gene under selection.

How the organization of genes on a chromosome shapes adaptation is essential for understanding evolutionary paths. Here, we investigate how adaptation to rapidly increasing levels of antibiotic depends on the chromosomal neighborhood of a drug-resistance gene inserted at different positions of the Escherichia coli chromosome. Using a dual-fluorescence reporter that allows us to distinguish gene amplifications from other up-mutations, we track in real-time adaptive changes in expression of the drug-resistance gene. We find that the relative contribution of several mutation types differs systematically between loci due to properties of neighboring genes: essentiality, expression, orientation, termination, and presence of duplicates. These properties determine rate and fitness effects of gene amplification, deletions, and mutations compromising transcriptional termination. Thus, the adaptive potential of a gene under selection is a system-property with a complex genetic basis that is specific for each chromosomal locus, and it can be inferred from detailed functional and genomic data.

On the mechanistic nature of epistasis in a canonical cis-regulatory element.

Understanding the relation between genotype and phenotype remains a major challenge. The difficulty of predicting individual mutation effects, and particularly the interactions between them, has prevented the development of a comprehensive theory that links genotypic changes to their phenotypic effects. We show that a general thermodynamic framework for gene regulation, based on a biophysical understanding of protein-DNA binding, accurately predicts the sign of epistasis in a canonical cis-regulatory element consisting of overlapping RNA polymerase and repressor binding sites. Sign and magnitude of individual mutation effects are sufficient to predict the sign of epistasis and its environmental dependence. Thus, the thermodynamic model offers the correct null prediction for epistasis between mutations across DNA-binding sites. Our results indicate that a predictive theory for the effects of cis-regulatory mutations is possible from first principles, as long as the essential molecular mechanisms and the constraints these impose on a biological system are accounted for.

Biased partitioning of the multidrug efflux pump AcrAB-TolC underlies long-lived phenotypic heterogeneity.

The molecular mechanisms underlying phenotypic variation in isogenic bacterial populations remain poorly understood. We report that AcrAB-TolC, the main multidrug efflux pump of Escherichia coli, exhibits a strong partitioning bias for old cell poles by a segregation mechanism that is mediated by ternary AcrAB-TolC complex formation. Mother cells inheriting old poles are phenotypically distinct and display increased drug efflux activity relative to daughters. Consequently, we find systematic and long-lived growth differences between mother and daughter cells in the presence of subinhibitory drug concentrations. A simple model for biased partitioning predicts a population structure of long-lived and highly heterogeneous phenotypes. This straightforward mechanism of generating sustained growth rate differences at subinhibitory antibiotic concentrations has implications for understanding the emergence of multidrug resistance in bacteria.

Bacterial flagella grow through an injection-diffusion mechanism.

The bacterial flagellum is a self-assembling nanomachine. The external flagellar filament, several times longer than a bacterial cell body, is made of a few tens of thousands subunits of a single protein: flagellin. A fundamental problem concerns the molecular mechanism of how the flagellum grows outside the cell, where no discernible energy source is available. Here, we monitored the dynamic assembly of individual flagella using in situ labelling and real-time immunostaining of elongating flagellar filaments. We report that the rate of flagellum growth, initially ∼1,700 amino acids per second, decreases with length and that the previously proposed chain mechanism does not contribute to the filament elongation dynamics. Inhibition of the proton motive force-dependent export apparatus revealed a major contribution of substrate injection in driving filament elongation. The combination of experimental and mathematical evidence demonstrates that a simple, injection-diffusion mechanism controls bacterial flagella growth outside the cell.

Intrinsic limits to gene regulation by global crosstalk.

Gene regulation relies on the specificity of transcription factor (TF)-DNA interactions. Limited specificity may lead to crosstalk: a regulatory state in which a gene is either incorrectly activated due to noncognate TF-DNA interactions or remains erroneously inactive. As each TF can have numerous interactions with noncognate cis-regulatory elements, crosstalk is inherently a global problem, yet has previously not been studied as such. We construct a theoretical framework to analyse the effects of global crosstalk on gene regulation. We find that crosstalk presents a significant challenge for organisms with low-specificity TFs, such as metazoans. Crosstalk is not easily mitigated by known regulatory schemes acting at equilibrium, including variants of cooperativity and combinatorial regulation. Our results suggest that crosstalk imposes a previously unexplored global constraint on the functioning and evolution of regulatory networks, which is qualitatively distinct from the known constraints that act at the level of individual gene regulatory elements.

Bacterial Autoimmunity Due to a Restriction-Modification System.

Restriction-modification (RM) systems represent a minimal and ubiquitous biological system of self/non-self discrimination in prokaryotes [1], which protects hosts from exogenous DNA [2]. The mechanism is based on the balance between methyltransferase (M) and cognate restriction endonuclease (R). M tags endogenous DNA as self by methylating short specific DNA sequences called restriction sites, whereas R recognizes unmethylated restriction sites as non-self and introduces a double-stranded DNA break [3]. Restriction sites are significantly underrepresented in prokaryotic genomes [4-7], suggesting that the discrimination mechanism is imperfect and occasionally leads to autoimmunity due to self-DNA cleavage (self-restriction) [8]. Furthermore, RM systems can promote DNA recombination [9] and contribute to genetic variation in microbial populations, thus facilitating adaptive evolution [10]. However, cleavage of self-DNA by RM systems as elements shaping prokaryotic genomes has not been directly detected, and its cause, frequency, and outcome are unknown. We quantify self-restriction caused by two RM systems of Escherichia coli and find that, in agreement with levels of restriction site avoidance, EcoRI, but not EcoRV, cleaves self-DNA at a measurable rate. Self-restriction is a stochastic process, which temporarily induces the SOS response, and is followed by DNA repair, maintaining cell viability. We find that RM systems with higher restriction efficiency against bacteriophage infections exhibit a higher rate of self-restriction, and that this rate can be further increased by stochastic imbalance between R and M. Our results identify molecular noise in RM systems as a factor shaping prokaryotic genomes.