Studies of the plasticity of transcription in Escherichia coli using single-molecule, in vivo detection techniques

The phenotypic characteristics of living organisms are shaped by the interactions of the genotype with the environment. In their lifetime, organisms are subject to various environmental changes, some of which are stressful. They cope up with these by means of phenotypic plasticity. In prokaryotes, this plasticity is achieved mostly by rapid adaptations of the gene expression profile. To better understand this, it is critical to study the mechanisms by which these adaptations are implemented. In Escherichia coli, transcription initiation is the first and most regulated step in gene expression. In vitro studies suggest that this is a complex, sequential process. Its rate-limiting steps regulate both the rate and the fluctuations of RNA production. These then determine the protein numbers and, thus, the cellular phenotype. In this work, we make use of state-of-the-art techniques in microscopy imaging, image processing and molecular probing to perform a quantitative analysis of the in vivo dynamics of transcription initiation in different environments in the prokaryotic model organism, E. coli. From the measurements, we characterize the plasticity of this process. For this, we used MS2-GFP fluorescent tagging of mRNA that allows detection of single mRNA molecules with confocal microscopy, shortly after their production. We also developed a tool to automatically track cell lineages in a time-lapse movie, and extract the spatiotemporal distribution of fluorescently tagged molecules in individual cells. From the analysis of the results, we show that, in vivo, the process of transcription initiation in E. coli is multi-stepped, as in vitro measurements had previously suggested. Also, the kinetics of each step can be independently controlled by different regulatory molecules. Further, the number and timing of the rate-limiting steps are affected by physiological changes that occur in cells when subject to changing environmental conditions. We conclude that the phenotypic plasticity of E. coli arises, partially, from the plasticity of the kinetics of the rate-limiting steps in transcription initiation

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Studies of the plasticity of transcription in Escherichia coli using single-molecule, in vivo detection techniques

The phenotypic characteristics of living organisms are shaped by the interactions of the genotype with the environment. In their lifetime, organisms are subject to various environmental changes, some of which are stressful. They cope up with these by means of phenotypic plasticity. In prokaryotes, this plasticity is achieved mostly by rapid adaptations of the gene expression profile. To better understand this, it is critical to study the mechanisms by which these adaptations are implemented. In Escherichia coli, transcription initiation is the first and most regulated step in gene expression. In vitro studies suggest that this is a complex, sequential process. Its rate-limiting steps regulate both the rate and the fluctuations of RNA production. These then determine the protein numbers and, thus, the cellular phenotype. In this work, we make use of state-of-the-art techniques in microscopy imaging, image processing and molecular probing to perform a quantitative analysis of the in vivo dynamics of transcription initiation in different environments in the prokaryotic model organism, E. coli. From the measurements, we characterize the plasticity of this process. For this, we used MS2-GFP fluorescent tagging of mRNA that allows detection of single mRNA molecules with confocal microscopy, shortly after their production. We also developed a tool to automatically track cell lineages in a time-lapse movie, and extract the spatiotemporal distribution of fluorescently tagged molecules in individual cells. From the analysis of the results, we show that, in vivo, the process of transcription initiation in E. coli is multi-stepped, as in vitro measurements had previously suggested. Also, the kinetics of each step can be independently controlled by different regulatory molecules. Further, the number and timing of the rate-limiting steps are affected by physiological changes that occur in cells when subject to changing environmental conditions. We conclude that the phenotypic plasticity of E. coli arises, partially, from the plasticity of the kinetics of the rate-limiting steps in transcription initiation

Design and Construction of a Synthetic Riboregulator-Based Platform for Metabolic Shunting of Pathways in <i>Lactococcus lactis</i>

Microbial cell factories are subjected to rewiring of basic metabolism to enhance the carbon flux towards the desired product pathway. Conventionally, this metabolic engineering approach often involves over-expression of pathway genes and knocking out genes in the competing pathways. However, these approaches result in severe metabolic burden and eventual poor performance of the cells. Particularly, where biomass formation and product synthesis depend on common precursors, permanent changes like knocking out genes will only result in poor titer. Hence, temporary decoupling of biomass formation and product synthesis is considered to be a potential alternative. In this study, we designed synthetic riboregulators, which are RNA-based genetic switches, to shunt metabolic pathways in Lactococcus lactis bacteria, a GRAS organism employed as a cell factory for many biocompounds. The riboregulators are then subjected to evaluation by tagging the cis-repressive sequence (crRNA) to mCherry, a fluorescent reporter and regulated by the constitutive promoter, [Formula: see text]. The trans-activating RNA (taRNA) that interacts with the crRNA is placed under the control of another inducible promoter, [Formula: see text]. First, we observed that when there is no induction of taRNA, there is negligible fluorescence of mCherry indicating the successful repression of translation by the cis-sequence as expected. This has been further verified by comparing this expression level with the expression level of [Formula: see text]-mCherry without the cis-sequence, using fluorometer. Results from this analysis suggest that there is [Formula: see text]% repression by the designed crRNA sequence. Next, we induced the cells with 2[Formula: see text]ng/mL of nisin in the mid-log phase. Upon induction, there is a maximum of three fold increase in the fluorescence levels when compared to the uninduced cells, suggesting that the trans-activation takes place inside the live cells. However, further studies are necessary to optimize the cis-trans ratio to achieve better dynamic range for expression modulation and time window for operating metabolic shunting of competing pathways successfully in L. lactis. </jats:p

In vivo transcription kinetics of a synthetic gene uninvolved in stress-response pathways in stressed Escherichia coli cells.

The fast adaptation of Escherichia coli to stressful environments includes the regulation of gene expression rates, mainly of transcription, by specific and global stress-response mechanisms. To study the effects of mechanisms acting on a global level, we observed with single molecule sensitivity the effects of mild acidic shift and oxidative stress on the in vivo transcription dynamics of a probe gene encoding an RNA target for MS2d-GFP, under the control of a synthetic promoter. After showing that this promoter is uninvolved in fast stress-response pathways, we compared its kinetics of transcript production under stress and in optimal conditions. We find that, following the application of either stress, the mean rates of transcription activation and of subsequent RNA production of the probe gene are reduced, particularly under oxidative stress. Meanwhile, the noise in RNA production decreases under oxidative stress, but not under acidic shift. From distributions of intervals between consecutive RNA productions, we infer that the number and duration of the rate-limiting steps in transcription initiation change, following the application of stress. These changes differ in the two stress conditions and are consistent with the changes in noise in RNA production. Overall, our measurements of the transcription initiation kinetics of the probe gene indicate that, following sub-lethal stresses, there are stress-specific changes in the dynamics of transcription initiation of the probe gene that affect its mean rate and noise of transcript production. Given the non-involvement of the probe gene in stress-response pathways, we suggest that these changes are caused by global response mechanisms of E. coli to stress

<i>In vivo</i> single-cell analysis using calcofluor - white staining detects high expression phenotype in <i>L. lactis</i> cultures engineered for hyaluronic acid production

ABSTRACTHyaluronic acid (HA) is a biopolymer with wide applications in the field of medicine and cosmetics. Bacterial production of HA has a huge market globally. Certain species of Streptococcus are native producers of HA but they are pathogenic. Therefore, safer organisms such as L. lactis are engineered for HA production. However, there are challenges such as low yield, low molecular weight and polydispersity of HA obtained from these cultures. Optimisation of bioprocess parameters and downstream purification parameters are being addressed to overcome these challenges. We explore these problems from the perspective of microbial heterogeneity, since variations in phenotype affect the yield and properties of the product in a bioreactor. For this perspective, a method to quantitatively assess the occurrence of heterogenous phenotypes depending on the amount of HA produced at the single-cell level is required. Here, we evaluated for the first time the use of calcofluor white staining method combined with in vivo fluorescence confocal microscopy to quantify the heterogeneity in phenotypes of L. lactis cells engineered for HA production.From the microscopy image analysis, we found that the population harbours significant heterogeneity with respect to HA production and our novel approach successfully differentiates these phenotypes. Using the fluorescence intensity levels, first we were able to confidently differentiate cells not expressing HA (Host cells without HA genes for expression) from cells with genes for HA production (GJP2) and induced for expression, as there is a consistently two-fold higher level of expression in the GJP2 cells independently of the cell size. Further, this method revealed the occurrence of two different phenotypes in GJP2 cultures, one of a high-expression phenotype (40% of the population) and the other one of a low-expression (remaining 60% of the population), and it is the high expression phenotype that contributes to the increase in the HA expression of the GJP2 population compared with the host cells. Thus, it is essential to identify the extrinsic and intrinsic factors that can favour most of the cells in the population to switch and stabilise into the high-expression phenotype state in a bioreactor, for higher yield and possibly reduced heterogeneity of the product, such as polydispersity in chain lengths. For such optimisation studies, this in vivo method serves as a promising tool for rapid detection of phenotypes in the bioreactor samples under varying conditions, allowing fine tuning of the factors to stabilise high-expression phenotypes thereby maximizing the yield.Graphical Abstractdone.Key PointsCalcofluor staining successfully differentiated the phenotypes based on HA levels.This study revealed the occurrence of significant heterogeneity in HA expression.This method will aid for rapid optimization of factors for improved HA production.</jats:sec

Quantification of the target gene mRNA copy number using an external calibration method.

<p>Error bars indicate the 95% confidence intervals of the mean of three separately calibrated experiments for each condition, assuming that the measurement error is Gaussian-like noise.</p