Effects of Intracellular and Partitioning Asymmetries in Escherichia coli

Cell divisions in Escherichia coli are, in general, morphologically symmetric. However, in a few cases, significant asymmetries between sister cells exist. These asymmetries between sister cells result in functional differences between them. For example, cells inheriting the older pole, over generations, accumulate more unwanted protein aggregates than their sister and, consequently, have a reduced growth rate. The reduced ability of these cells to reproduce shows that even these unicellular organisms are susceptible to the effects of aging. To understand senescence in these organisms, it is critical to investigate the sources as well as the functional consequences of asymmetries in division.In this thesis, we characterize mechanisms responsible for functional and morphological asymmetries in division in E. coli cells, using live, single-cell, single-molecule imaging techniques and detailed stochastic models. First, to understand the functional asymmetries due to the heterogeneous spatial distribution of large, inert protein complexes, we study the kinetics of segregation and retention of such complexes by observing these events, one event at a time. For that, we track individual MS2-GFP tagged RNA complexes, as they move in the cell cytoplasm, and characterize the mechanisms responsible for their long-term spatial distribution and resulting partitioning. Next, to understand the morphological asymmetries, we study the difference in cell sizes between sister cells at division under different environmental conditions. Finally, we present the models and simulators developed to characterize and mimic these processes, as well as to explore their functional consequences.Our results suggest that functional and morphological asymmetries in division, in the growth conditions studied, appear to be mostly driven by the nucleoid. In particular, we find that the fluorescent complexes are retained at the poles due to nucleoid occlusion. Further, the positioning of the point of division is also regulated by the degree of proximity between the two replicated nucleoids in the cell at the moment preceding division. Finally, based on simulation results of the models in extreme conditions, we suggest that asymmetries in these processes in division can enhance the mean vitality of E. coli cell populations. Overall, the results suggest that nucleoid occlusion contributes, in different ways, to heterogeneities in E. coli cells that ultimately generate phenotypic differences between sister cells

Characterization of population heterogeneity in a model biotechnological process using Pseudomonas putida

Biotechnological processes are distinguished from classical chemistry by employing bio-molecules or whole cells as the catalytic element, providing unique reaction mechanisms with unsurpassed specificity. Whole cells are the most versatile \''factories\'' for natural or non-natural products, however, the conversion of e.g. hydrophobic substrates can quickly become cytotoxic. One host organism with the potential to handle such conditions is the gram-negative bacterium Pseudomonas putida, which distinguishes itself by solvent tolerance, metabolic flexibility, and genetic amenability. However, whole cell bioconversions are highly complex processes. A typical bottleneck compared to classical chemistry is lower yield and reproducibility owing to cell-to-cell variability. The intention of this work was therefore to characterize a model producer strain of P. putida KT2440 on the single cell level to identify non-productive or impaired subpopulations. Flow cytometry was used in this work to discriminate subpopulations regarding DNA content or productivity, and further mass spectrometry or digital PCR was employed to reveal differences in protein composition or plasmid copy number. Remarkably, productivity of the population was generally bimodally distributed comprising low and highly producing cells. When these two subpopulations were analyzed by mass spectrometry, only few metabolic changes but fundamental differences in stress related proteins were found. As the source for heterogeneity remained elusive, it was hypothesized that cell cycle state may be related to production capacity of the cells. However, subpopulations of one, two, or higher fold DNA content were virtually identical providing no clear hints for regulatory differences. On the quest for heterogeneity the loss of genetic information came into focus. A new work flow using digital PCR was created to determine the absolute number of DNA copies per cell and, finally, lack of expression could be attributed to loss of plasmid in non-producing cells. The average plasmid copy number was shown to be much lower than expected (1 instead of 10-20). In conclusion, this work established techniques for the quantification of proteins and DNA in sorted subpopulations, and by these means provided a highly detailed picture of heterogeneity in a microbial population

Dynamics of Genetic Circuits with Molecule Partitioning Errors in Cell Division and RNA-RNA Interactions

Many signaling and regulatory molecules within cells exist in very few copies per cell. Any process affecting even limited numbers of these molecules therefore has the potential to affect the dynamics of the biochemical networks of which they are a part. This sensitivity to small copy-number changes is what allows stochasticity in gene expression to introduce a degree of randomness in what cells do. While this randomness can be suppressed, it does not appear to be so in many biological systems, at least not to the maximum degree possible. This suggests that this randomness is not necessarily detrimental to cell populations, as it can produce qualitatively new behaviours in genetic networks which may be utilized by cells.In this thesis, two other mechanisms are investigated which, through their interaction with low copy-number molecules, are able to produce qualitatively different dynamics in genetic networks: the stochastic partitioning of molecules in cell division, and the direct interaction of two low copy-number molecules. For this, a novel simulator of chemical kinetics is first presented, designed to simulate the dynamics of genetic circuits inside growing populations of cells. It is then used to study a genetic switch where one repressive link is formed by direct interaction between RNA molecules. This arrangement was found to decouple the stability of the two noisy attractors of the network and the speeds of the state transitions. In other words, it allows the network to have two equally-stable noisy attractors, but differing state transition speeds.Next, the cell-to-cell diversity in RNA numbers (as quantified by the normalized variance) of a single gene over time in a growing model cell population was studied as a function of the division synchrony. In the model, synchronous cell divisions introduce transient increases in the cell-to-cell diversity in RNA numbers of the population, a prediction which was verified using single-molecule measurements of RNA numbers. Finally, the effects of the stochastic partitioning of regulatory molecules in cell division on the dynamics of two genetic circuits, a switch and a clock, were studied. Of these two circuits, the switch has the most dramatic changes in its dynamics, brought on by the inevitable negative correlation in molecule numbers that sister cells inherit. This negative correlation can allow a cell population to partition the phenotypes of the individual cells with less variance than a binomial distribution.These results advance our understanding of the different behaviours that can be produced in genetic circuits due to these two mechanisms. Since they produce unique behaviours, these mechanisms, and combinations thereof, are expected to be used for specialized purposes in natural genetic circuits. Further, since the downstream effects of these mechanisms may be more predictable than, e.g., modifying promoter sequences, they may also be useful in the design and implementation of future synthetic genetic circuits with specific behaviours.<br/

\u3cem\u3ePseudomonas aeruginosa\u3c/em\u3e reverse diauxie is a multidimensionl, optimized, resource utilization strategy

Pseudomonas aeruginosa is a globally-distributed bacterium often found in medical infections. The opportunistic pathogen uses a different, carbon catabolite repression (CCR) strategy than many, model microorganisms. It does not utilize a classic diauxie phenotype, nor does it follow common systems biology assumptions including preferential consumption of glucose with an ‘overflow’ metabolism. Despite these contradictions, P. aeruginosa is competitive in many, disparate environments underscoring knowledge gaps in microbial ecology and systems biology. Physiological, omics, and in silico analyses were used to quantify the P. aeruginosa CCR strategy known as ‘reverse diauxie’. An ecological basis of reverse diauxie was identified using a genome-scale, metabolic model interrogated with in vitro omics data. Reverse diauxie preference for lower energy, nonfermentable carbon sources, such as acetate or succinate over glucose, was predicted using a multidimensional strategy which minimized resource investment into central metabolism while completely oxidizing substrates. Application of a common, in silico optimization criterion, which maximizes growth rate, did not predict the reverse diauxie phenotypes. This study quantifies P. aeruginosa metabolic strategies foundational to its wide distribution and virulence including its potentially, mutualistic interactions with microorganisms found commonly in the environment and in medical infections

Efficiency and Robustness to Nonoptimal Temperatures of Nucleoid Exclusion Processes in Escherichia coli

Prokaryotic organisms, such as Escherichia coli, lack internal wall. Nevertheless, they have internal spatial organization, which is a critical requirement for the proper functioning of several cellular processes. One of the internal structures that is crucial for the emergence and maintenance of such organization is the nucleoid. With a higher density than the cytoplasm, the nucleoid is able to segregate different macromolecules — such as protein aggregates and chemotaxis clusters — to the cell poles, while also placing structures, such as the Z-ring, between recently replicated nucleoids during cell division. In this regard, for a cell population to thrive in fluctuating environments, these cellular processes need not only to be efficient in optimal conditions, but also robust to nonoptimal conditions. Here, we study the efficiency of the processes of segregation of protein aggregates, polarization of chemotaxis network, and Z-ring positioning at midcell and relate it with the nucleoid’s morphology. In order to evaluate their robustness to nonoptimal conditions, we also study the effects of temperature shifts on the nucleoid(s) morphology, and how this then affects the efficiency of these biophysical processes. For this, we collected confocal microscopy images of populations of cells with fluorescently tagged protein aggregates, protein clusters composing the chemotaxis networks, and FtsZ-proteins composing Z-rings, at different temperatures. In addition, we also stained the nucleoids of these cells. From the analysis of the data collected from the images, we found that, for each temperature condition, the spatial distribution of the cellular components observed is consistent with the nucleoid’s volume exclusion effect. Furthermore, we found that the nucleoid’s length along the major cell axis is correlated with the kurtosis of the spatial distribution of protein aggregates and chemotaxis clusters along that axis. Similarly, the distribution of distances between replicated nucleoids (prior to cell division) along the major cell axis is correlated with the kurtosis of the spatial distribution of Z-rings along that axis. Finally, we found a negative correlation between the efficiency of these spatial placement processes at optimal temperatures and their robustness to nonoptimal temperatures, indicating a trade-off between these features. Overall, these results suggest that the robustness of the morphological features of the nucleoid to temperature shifts contribute to the adaptability of E. coli to non-optimal temperatures

Stochastic Processes as a Source of Cell to Cell Diversity and Cellular Ageing

Even populations of monoclonal cells exhibit phenotypic diversity. There are several sources generating such diversity, including stochasticity in the dynamics of gene expression, and the stochastic partitioning of molecules during division. This thesis focuses on the construction and simulation of a realistic model of gene expression and on the stochastic partitioning of cellular components during cell division. First, we present and make use of statistical methods to extract information on the kinetics of gene expression from live-cell measurements at the single RNA molecule level. This information allows us to characterize the kinetics of the multi-stepped process of transcription initiation, including the degree of noise in transcript production, as well as the kinetics of partitioning of protein aggregates by the cell’s poles. A model of single gene expression in a growing population of cells and a model of ageing in bacteria are then constructed based upon these measurements. Next, we present a new simulator which uses the Stochastic Simulation Algorithm to simulate the dynamics of intracellular processes in populations of cells, each of which able to grow and divide with random partitioning of molecules. Cells are represented in the simulator by compartments that can be created and destroyed at runtime. Logarithmic simulation algorithms and efficient data structures were designed and are here presented, which minimize the computational cost of simulating the dynamics of large cell populations that involve a large number of chemical reactions

The Role of Nucleoid Exclusion in the Intracellular Spatial Organization of Escherichia coli

Not long ago, bacterial cells were regarded as organisms with hardly any internal organization, due to lack of visible physical compartments and, thus, proteins were believed to be distributed randomly. Since then, advances in microscopy, in in vivo protein labeling with fluorescent tags, and in image analysis techniques have enabled us to probe biological events at a single-cell, singletime moment, and single-molecule level. The results from these observations have led to a radical change in this view and, thus, revolutionized the field of bacterial cell biology. Namely, this novel source of information has made evident that proper bacterial functioning is not possible without a highly spatially organized, dynamic internal composition that depends on the deployment of functional proteins and other cellular components in specific locations, at specific moments.The spatiotemporal organization of the functional proteins and other cellular components play a fundamental role in several key regulatory processes, such as transcription, translation and cell division. One class of proteins, termed as ‘DNA-binding proteins’, are associated with DNA replication and segregation. Not surprisingly, they preferentially locate at midcell, where the chromosomal DNA is condensed into a dynamic structure called ‘nucleoid’. Another class of proteins, termed as ‘polar proteins’, are majorly involved in physiological behaviors such as chemotaxis, sugar uptake, motility and adhesion. In agreement, they are preferentially localized at the cell poles in the case of rod-shaped bacteria such as E. coli. Finally, there is a third class of proteins, called as ‘cytoskeletal proteins’, whose location differ widely during cell growth. For example, the Min system, a major cell division regulatory system, consisting of MinCDE proteins have a remarkable dynamic pattern inside the cell. These proteins localize for about half a minute in one cell half and then switch rapidly to the opposite half. This back and forth motion continues until the polymerization of the division protein FtsZ results in a ring-like structure at the cell center prior to cell division.Cellular components, other than functional proteins, also exhibit a highly-organized spatial distribution. These components include plasmids, enzyme megacomplexes and unwanted protein aggregates. For example, protein aggregates, formed as a result of environment stress or errors in protein homeostasis, are generally sequestered into inclusion bodies (IBs) that localize at the cell poles. This process of polar localization is symmetric. However, following several cell division events, results in progeny cells containing the old pole having more aggregates than the new pole possessing progeny cells. Subsequent divisions lead to cell generations where some cells inherit more aggregates than others. Importantly, this was found to be positively correlated with increased division times, i.e., cellular aging. It is believed that such asymmetric partitioning of unwanted aggregates may be critical for the rejuvenation of bacterial populations. It is thus of major importance to understand the underlying mechanisms that are responsible for the above-described events.In this thesis, using Escherichia coli as our model organism, we started by investigating and validating the hypothesis that the presence of the nucleoid at the midcell is responsible for the ability of this organism to segregate unwanted protein aggregates to the cell poles. We next investigated and characterized the robustness of these mechanisms to external perturbations and stressful environmental conditions. Afterwards, we hypothesized that the phenomenon of nucleoid exclusion should not be limited to protein aggregates alone but, instead, for physical reasons, it should influence any large macromolecule that is not affected by a transport or self-propelling mechanism (which is the case of all proteins in E. coli). Consequently, we hypothesized and subsequently proved that it should influence self-assembling proteins, such as the transmembrane Tsr chemoreceptors, which have a major role in bacterial chemotaxis. In addition, we also investigated to what extent cell-to-cell diversity in nucleoid sizes contributes to the cell-to-cell diversity in the spatial distribution of polar-localized proteins. For these studies, we made use of efficient fluorescent tags, in vivo single-cell, single-molecule time-lapse microscopy, tailored image and signal processing techniques and stochastic biophysical models.Our results provide new perspectives regarding the role of the nucleoid in the spatial organization of protein aggregates as well as chemoreceptor clusters in E. coli. Interestingly, regarding the latter, nucleoid exclusion from midcell was shown not to be the sole phenomenon for the proper localization of Tsr protein clusters. However, it is expected to be the most robust, namely, in stressful environments or when the cell is subject to external perturbations, than the diffusion-andcapture mechanism mediated by the Tol-Pal complexes, as it does not require production of proteins or is under stringent control. Further, unlike the other mechanism, it is energy-free.Given the rapid developments in single-cell biology techniques, particularly the emergence of super-resolution microscopy techniques, improved fluorescent probes, high-throughput and largescale biochemical methods and theoretical tools, we expect several developments in the near future that will allow assessing further the role of the nucleoid as a ‘spatial organizer’ of the cellular architecture of E. coli

Environment-sensing Mechanisms of Gene Expression and their Effects on the Dynamics of Genetic Circuits across Cell Generations

In genetic circuits, the constituent genes do not interact only between themselves, they are also affected by regulatory molecules of the host cells that support the circuits’ operation and by the environmental conditions. These factors, along with the intrinsic noise in gene expression, affect the functioning of the circuits. As such, to understand the structure of natural circuits and to engineer functional synthetic circuits, one needs to characterize thoroughly how external factors and perturbations from the environment may affect their behavior.This thesis focused on two cellular mechanisms through which the dynamics of gene expression becomes environment dependent: the intake of gene expression regulatory molecules from the media and the σ factor competition. The first mechanism determines the dynamics by which inducer molecules in the media enter the cell cytoplasm and trigger or repress the expression of the target gene. The second mechanism allows cells to change its gene expression profile to adapt to specific stress conditions.Following the characterization of the effects of these mechanisms on the expression dynamics of individual genes from live, single cell measurements, we then performed in silico assessments on how these effects at the single gene level propagate to the circuit level. Here, the dynamics of genetic circuits was observed in both non-dividing and dividing cell populations, where errors in the partitioning of molecules in cell division occur and introduce significant variance between sister cells.From these studies, with the knowledge on the factors of the host cells and their environment sensing mechanisms, more predictive models of the circuits’ dynamics are expected to emerge. The models would further help in identifying what circuit composition, properties of the host strains and environmental conditions are needed for the circuits to exhibit the desired behavior