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1.  Localization of Protein Aggregation in Escherichia coli Is Governed by Diffusion and Nucleoid Macromolecular Crowding Effect 
PLoS Computational Biology  2013;9(4):e1003038.
Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results provide evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3D individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion-aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of “soft” intracellular structuring (based on macromolecular crowding) in diffusion-based protein localization in E. coli.
Author Summary
Localization of proteins to specific positions inside bacteria is crucial to several physiological processes, including chromosome organization, chemotaxis or cell division. Since bacterial cells do not possess internal sub-compartments (e.g., cell organelles) nor vesicle-based sorting systems, protein localization in bacteria must rely on alternative mechanisms. In many instances, the nature of these mechanisms remains to be elucidated. In Escherichia coli, the localization of aggregates of misfolded proteins at the poles or the center of the cell has recently been linked to aging. However, the molecular mechanisms governing this localization of the protein aggregates remain controversial. To identify these mechanisms, we have devised an integrated strategy combining innovative experimental and modeling approaches. Our results show the importance of the increased macromolecular crowding in the nucleoids, the regions within the cell where the bacterial chromosome preferentially condensates. They indicate that a purely diffusive pattern of aggregates mobility combined with nucleoid occlusion underlies their accumulation in polar and mid-cell positions.
PMCID: PMC3636022  PMID: 23633942
2.  Pre-Disposition and Epigenetics Govern Variation in Bacterial Survival upon Stress 
PLoS Genetics  2012;8(12):e1003148.
Bacteria suffer various stresses in their unpredictable environment. In response, clonal populations may exhibit cell-to-cell variation, hypothetically to maximize their survival. The origins, propagation, and consequences of this variability remain poorly understood. Variability persists through cell division events, yet detailed lineage information for individual stress-response phenotypes is scarce. This work combines time-lapse microscopy and microfluidics to uniformly manipulate the environmental changes experienced by clonal bacteria. We quantify the growth rates and RpoH-driven heat-shock responses of individual Escherichia coli within their lineage context, stressed by low streptomycin concentrations. We observe an increased variation in phenotypes, as different as survival from death, that can be traced to asymmetric division events occurring prior to stress induction. Epigenetic inheritance contributes to the propagation of the observed phenotypic variation, resulting in three-fold increase of the RpoH-driven expression autocorrelation time following stress induction. We propose that the increased permeability of streptomycin-stressed cells serves as a positive feedback loop underlying this epigenetic effect. Our results suggest that stochasticity, pre-disposition, and epigenetic effects are at the source of stress-induced variability. Unlike in a bet-hedging strategy, we observe that cells with a higher investment in maintenance, measured as the basal RpoH transcriptional activity prior to antibiotic treatment, are more likely to give rise to stressed, frail progeny.
Author Summary
Individual organisms of identical genetic background, living in a homogeneous constant environment, may nonetheless exhibit observable differences dubbed phenotypic plasticity or variability. When such a population is challenged with an unforeseen stress, the disparity among individuals may increase, yielding different strategies in response. This work addresses the occurrence and propagation of phenotypic variation as it affects bacterial survival in response to mild antibiotic treatments. We recorded images of single bacterial cells as they divide prior to and during exposure to a sub-lethal level of streptomycin, a ribosome-targeted antibiotic. We found that individual differences increase upon stress to the extent that cells may either die or survive the treatment. Differentiation events were traced back prior to exposure. We suggest that a positive feedback loop, governed by increased membrane permeability, underlies the transient cell memory observed. Cells with relatively high basal stress-response levels prior to stress are not primed for better survival, but are rather more likely to succumb to antibiotic treatment. As pathogens commonly encounter sub-lethal doses of antibiotics, their survival may be better understood in light of this study.
PMCID: PMC3527273  PMID: 23284305
3.  Evidence for an evolutionary antagonism between Mrr and Type III modification systems 
Nucleic Acids Research  2011;39(14):5991-6001.
The Mrr protein of Escherichia coli is a laterally acquired Type IV restriction endonuclease with specificity for methylated DNA. While Mrr nuclease activity can be elicited by high-pressure stress in E. coli MG1655, its (over)expression per se does not confer any obvious toxicity. In this study, however, we discovered that Mrr of E. coli MG1655 causes distinct genotoxicity when expressed in Salmonella typhimurium LT2. Genetic screening enabled us to contribute this toxicity entirely to the presence of the endogenous Type III restriction modification system (StyLTI) of S. typhimurium LT2. The StyLTI system consists of the Mod DNA methyltransferase and the Res restriction endonuclease, and we revealed that expression of the LT2 mod gene was sufficient to trigger Mrr activity in E. coli MG1655. Moreover, we could demonstrate that horizontal acquisition of the MG1655 mrr locus can drive the loss of endogenous Mod functionality present in S. typhimurium LT2 and E. coli ED1a, and observed a strong anti-correlation between close homologues of MG1655 mrr and LT2 mod in the genome database. This apparent evolutionary antagonism is further discussed in the light of a possible role for Mrr as defense mechanism against the establishment of epigenetic regulation by foreign DNA methyltransferases.
PMCID: PMC3152355  PMID: 21504983
4.  Emergence of Variability in Isogenic Escherichia coli Populations Infected by a Filamentous Virus 
PLoS ONE  2010;5(7):e11823.
The spread of epidemics not only depends on the average number of parasites produced per host, but also on the existence of highly infectious individuals. It is widely accepted that infectiousness depends on genetic and environmental determinants. However, even in clonal populations of host and viruses growing in homogeneous conditions, high variability can exist. Here we show that Escherichia coli cells commonly display high differentials in viral burst size, and address the kinetics of emergence of such variability with the non-lytic filamentous virus M13. By single-cell imaging of a virally-encoded fluorescent reporter, we monitor the viral charge distribution in infected bacterial populations at different time following infection. A mathematical model assuming autocatalytic virus replication and inheritance of bacterial growth rates quantitatively reproduces the experimental distributions, demonstrating that deterministic amplification of small host inhomogeneities is a mechanism sufficient to explain large and highly skewed distributions. This mechanism of amplification is general and may occur whenever a parasite has an initial phase of exponential growth within its host. Moreover, it naturally reproduces the shift towards higher virulence when the host is experimenting poor conditions, as observed commonly in host-parasite systems.
PMCID: PMC2910729  PMID: 20676396
5.  Pre-dispositions and epigenetic inheritance in the Escherichia coli lactose operon bistable switch 
Under conditions of bistable induction of Escherichia coli lac operon, epigenetic patterns of sublineages of ‘on' and ‘off' cells originate from distinguishable ancestors up to two generations before induction.We found two switching pre-disposing factors, namely low repressor levels and slow growth, demonstrating that stochasticity in gene expression and global physiology synergistically determine the single-cell responses.A quantitative model where growth rate acts through simple dilution of intracellular content and repressor level controls the basal activity of the operon demonstrates that both growth rate and repressor concentration influence the cell switching ability.
The bacterium Escherichia coli, like many other microorganisms can use different sugars as a carbon source and uses some of these sugars in preference to others. For example, when grown in the presence of both lactose and glucose, the bacteria first consume glucose and use lactose only when glucose is exhausted. To this end, the enzymes necessary for lactose uptake and metabolism, grouped in one transcriptional unit called the lac operon (lacZ, Y, A encoding for the lactose degrading enzyme (β-galactosidase), permease and transacetylase, respectively) are produced only in the absence of glucose and in the presence of lactose or its analogs, such as the non-metabolizable analog thiomethyl-β-galactoside (TMG). In the absence of such inducers, the transcription of the operon is inhibited by the repressor molecule LacI. This inhibition is relieved by the inducers, which bind and inactivate LacI, initiating an amplifying feedback loop through the expression of the permease that ensures a high influx of inducer to maintain the operon's expression in the ‘on' state. This phenomenon of adaptive enzyme production has been widely studied since its discovery by Jacques Monod and François Jacob and is one of the most famous and best characterized examples of transcriptional gene regulation. E. coli lactose operon is also a paradigm of cellular differentiation. Indeed, in the presence of an intermediate concentration of TMG, an isogenic bacterial population is divided in two subpopulations of cells with the operon's genes either turned on or remaining off. The differentiation step is generally hypothesized to depend on fluctuations in expression of the operon's proteins. Nevertheless, it is still poorly characterized. On the basis of experimental and theoretical approaches, we explored the determinants of cell fate in this system.
We designed a microfluidic device allowing the observation of single cells growing within a microcolony under conditions that can be changed at will. We used this setup to study phenotypic variability in the lactose operon induction under conditions leading to a transient bimodality of lac expression in the population. We used an E. coli strain modified to express the yellow fluorescent protein (YFP) and the cyan fluorescent protein (CFP), both under the control of a promoter regulated by LacI (PLlacO1). Therefore, yellow and cyan fluorescence intensities both represent the concentration of active repressor molecules and indirectly, the expression state of the lactose operon. Microcolonies originating from a single cell were grown in the microfluidic device and followed by time-lapse microscopy. During the first generations of growth, cells were grown in the absence (or with a very low concentration) of inducer and after several generations, TMG was introduced at intermediate concenteration into the medium and maintained thereafter. In the absence of TMG, cells exhibit an overall weak fluorescence yet with significant variations between cells that were shown to correspond in part to the variability in the intracellular concentration of active LacI molecules. Upon induction, transient bimodality is observed, as the cells are divided between two subpopulations of bright and dim fluorescence.
We found a strong clustering of induced cells within their genealogical trees, indicating a substantial epigenetic inheritance. This epigenetic inheritance can be traced back up to two generations prior induction, suggesting that some molecular determinants of cell fate are epigenetically inherited with a short-range memory lasting around two divisions.
The promoter used to control fluorescence proteins expression is sensitive to small variations in active LacI concentration. Thus, in the absence of inducer, these variations result into correlated variations of YFP and CFP levels. We used the arithmetical mean of yellow and cyan fluorescence intensities to estimate the concentration of active LacI in the cells. We found that the cells exhibiting a low LacI concentration before induction are more likely to be induced upon TMG introduction. Likewise, the slowly growing cells were found to have a higher switching probability than the fast-growing ones. We used a multivariate analysis based on a generalized linear model to estimate the correlations of pre-induction LacI concentration and growth rate with the switching probability (Figure 5C). This analysis confirms that both LacI concentration and growth rate are correlated with the switching probability and demonstrates that even though LacI concentration and growth rate can be linked, their correlations with the switching probability represent independent effects. Together, these effects can account for 90% of the observed switching events.
To gain a better understanding of the possible influence of LacI expression fluctuations and growth rate on the switching probability of a cell, we used a model consisting in a system of differential equations and describing the dynamics of the lactose utilization network. In this model, LacI concentration controls the basal level of expression of the operon and the growth rate acts through the dilution of intracellular components. According to this model, depending on both LacI concentration and growth rate, a cell can be in a monostable or bistable regime. Therefore, monostable and bistable cells can coexist in the population due to parameters' variability. In addition, for cells in the bistable regime, the size of the minimal LacY burst necessary to trigger induction increases with LacI concentration and growth rate. Thus, in agreement with our experimental results, these two variables control the sensitivity of the cell to permease bursts and therefore influence its switching probability.
We thus found pre-disposing factors governing the lactose operon switching in a regime of transient bimodality. Some factors, such as LacI and LacY concentrations result from stochasticity at the local level of the network. On the contrary, growth rate variability represents variations in the cell global physiology. Therefore, the effects of local stochasticity are coupled with the influence of the global physiology, demonstrating the importance of considering the embedding of a particular genetic network in the whole cellular physiology to understand fully its dynamics.
The lactose operon regulation in Escherichia coli is a primary model of phenotypic switching, reminiscent of cell fate determination in higher organisms. Under conditions of bistability, an isogenic cell population partitions into two subpopulations, with the operon's genes turned on or remaining off. It is generally hypothesized that the final state of a cell depends solely on stochastic fluctuations of the network's protein concentrations, particularly on bursts of lactose permease expression. Nevertheless, the mechanisms underlying the cell switching decision are not fully understood. We designed a microfluidic system to follow the formation of a transiently bimodal population within growing microcolonies. The analysis of genealogy and cell history revealed the existence of pre-disposing factors for switching that are epigenetically inherited. Both the pre-induction expression stochasticity of the lactose operon repressor LacI and the cellular growth rate are predictive factors of the cell's response upon induction, with low LacI concentration and slow growth correlating with higher switching probability. Thus, stochasticity at the local level of the network and global physiology are synergistically involved in cell response determination.
PMCID: PMC2872608  PMID: 20393577
adaptation; bistability; differentiation; lac operon; stochastic gene expression
6.  Mutations in two global regulators lower individual mortality in Escherichia coli 
Molecular Microbiology  2007;67(1):2-14.
There has been considerable investigation into the survival of bacterial cells under stress conditions, but little is known about the causes of mortality in the absence of exogenous stress. That there is a basal frequency of cell death in such populations may reflect that it is either impossible to avoid all lethal events, or alternatively, that it is too costly. Here, through a genetic screen in the model organism Escherichia coli, we identify two mutants with lower frequencies of mortality: rssB and fliA. Intriguingly, these two genes both affect the levels of different sigma factors within the cell. The rssB mutant displays enhanced resistance to multiple external stresses, possibly indicating that the cell gains its increased vitality through elevated resistance to spontaneous, endogenous stresses. The loss of fliA does not result in elevated stress resistance; rather, its survival is apparently due to a decreased physical stress linked to the insertion of the flagellum through the membrane and energy saved through the loss of the motor proteins. The identification of these two mutants implies that reducing mortality is not impossible; rather, due to its cost, it is subject to trade-offs with other traits that contribute to the competitive success of the organism.
PMCID: PMC2229837  PMID: 18036141
7.  Dissecting the Genetic Components of Adaptation of Escherichia coli to the Mouse Gut 
PLoS Genetics  2008;4(1):e2.
While pleiotropic adaptive mutations are thought to be central for evolution, little is known on the downstream molecular effects allowing adaptation to complex ecologically relevant environments. Here we show that Escherichia coli MG1655 adapts rapidly to the intestine of germ-free mice by single point mutations in EnvZ/OmpR two-component signal transduction system, which controls more than 100 genes. The selective advantage conferred by the mutations that modulate EnvZ/OmpR activities was the result of their independent and additive effects on flagellin expression and permeability. These results obtained in vivo thus suggest that global regulators may have evolved to coordinate activities that need to be fine-tuned simultaneously during adaptation to complex environments and that mutations in such regulators permit adjustment of the boundaries of physiological adaptation when switching between two very distinct environments.
Author Summary
The mammalian intestine is a privileged physiological site to study how coevolution between hosts and the trillions of bacteria present in the microbiota has shaped the genome of each partner and promoted the development of mutualistic interactions. Herein we have used germ-free mice, a simplified albeit ecologically relevant system, to analyse intestinal adaptation of a model bacterial strain, Escherichia coli MG1655. Our results show that single point mutations in the ompB master regulator confer a striking selective adaptive advantage. OmpB comprises EnvZ, a transmembrane sensor with a dual kinase/phosphatase activity, and OmpR, a transcription factor controlling more than 100 target genes. In response to environmental changes, EnvZ modulates the phosphorylation and thereby the transcriptional activity of OmpR. We further show that the selective advantage conferred by OmpB mutations is related to their additive and independent effects on genes regulating permeability and flagellin expression, two major set of genes controlled by OmpR. These results suggest that global regulators may have evolved to coordinate physiological activities necessary for adaptation to complex environments and that mutations offer a complementary genetic mechanism to adjust the scale of the physiological regulation controlled by these regulators in distinct environments.
PMCID: PMC2174974  PMID: 18193944
8.  Construction of a multiple fluorescence labelling system for use in co-invasion studies of Listeria monocytogenes 
BMC Microbiology  2006;6:86.
Existing virulence models are often difficult to apply for quantitative comparison of invasion potentials of Listeria monocytogenes. Well-to-well variation between cell-line based in vitro assays is practically unavoidable, and variation between individual animals is the cause of large deviations in the observed capacity for infection when animal models are used.
One way to circumvent this problem is to carry out virulence studies as competition assays between 2 or more strains. This, however, requires invasion-neutral markers that enable easy discrimination between the different strains.
A fluorescent marker system, allowing visualization and identification of single L. monocytogenes cells as well as colonies in a non-destructive manner, was developed. Five different fluorescent labels are available, and allowed simultaneous visual discrimination between three differently labelled strains at the single cell level by use of fluorescence microscopy. More than 90% of the L. monocytogenes host cells maintained the fluorescence tags for 40 generations.
The fluorescence tags did not alter the invasive capacity of the L. monocytogenes cells in a traditional Caco-2 cell invasion assay, and visual discrimination between invaded bacteria carrying different fluorescent labels inside the cells was possible.
The constructed fluorescent marker system is stable, easy to use, does not affect the virulence of L. monocytogenes in Caco-2 cell assays, and allows discrimination between differently labelled bacteria after internalization in these cells.
PMCID: PMC1599739  PMID: 17014739

Results 1-8 (8)