In Escherichia coli , tetracycline prevents translation. When subject to tetracycline, E. coli express TetA to pump it out by a mechanism that is sensitive, while fairly independent of cellular metabolism. We constructed a target gene, P tetA -mRFP1-96BS, with a 96 MS2-GFP binding site array in a single-copy BAC vector, whose expression is controlled by the tetA promoter. We measured the in vivo kinetics of production of individual RNA molecules of the target gene as a function of inducer concentration and temperature. From the distributions of intervals between transcription events, we find that RNA production by P tetA is a sub-Poissonian process. Next, we infer the number and duration of the prominent sequential steps in transcription initiation by maximum likelihood estimation. Under full induction and at optimal temperature, we observe three major steps. We find that the kinetics of RNA production under the control of P tetA , including number and duration of the steps, varies with induction strength and temperature. The results are supported by a set of logical pairwise Kolmogorov-Smirnov tests. We conclude that the expression of TetA is controlled by a sequential mechanism that is robust, whereas sensitive to external signals.

a b s t r a c tThe kinetics of transcription initiation in Escherichia coli depend on the duration of two rate-limiting steps, the closed and the open complex formation. In a lac promoter variant, P lac/ara-1 , the kinetics of these steps is controlled by IPTG and arabinose. From in vivo single-RNA measurements, we find that induction affects the mean and normalized variance of the intervals between consecutive RNA productions. Transcript production is sub-Poissonian in all conditions tested. The kinetics of each step is independently controlled by a different inducer. We conclude that the regulatory mechanism of P lac/ara-1 allows the stochasticity of gene expression to be environment-dependent.

Motivation: Cell division in Escherichia coli is morphologically symmetric. However, as unwanted protein aggregates are segregated to the cell poles and, after divisions, accumulate at older poles, generate asymmetries in sister cells' vitality. Novel single-molecule detection techniques allow observing aging-related processes in vivo, over multiple generations, informing on the underlying mechanisms. Results: CellAging is a tool to automatically extract information on polar segregation and partitioning in division of aggregates in E.coli, and on cellular vitality. From time-lapse, parallel brightfield and fluorescence microscopy images, it performs cell segmentation, alignment of brightfield and fluorescence images, lineage construction and pole age determination, and it computes aging-related features. We exemplify its use by analyzing spatial distributions of fluorescent protein aggregates from images of cells across generations. Availability: CellAging, instructions and an example are available at

The morphological symmetry of the division process of Escherichia coli is well-known. Recent studies verified that, in optimal growth conditions, most divisions are symmetric, although there are exceptions. We investigate whether such morphological asymmetries in division introduce functional asymmetries between sister cells, and assess the robustness of the symmetry in division to mild chemical stresses and sub-optimal temperatures. First, we show that the difference in size between daughter cells at birth is positively correlated to the difference between the numbers of fluorescent protein complexes inherited from the parent cell. Next, we show that the degree of symmetry in division observed in optimal conditions is robust to mild acidic shift and to mild oxidative stress, but not to sub-optimal temperatures, in that the variance of the difference between the sizes of sister cells at birth is minimized at 37 °C. This increased variance affects the functionality of the cells in that, at sub-optimal temperatures, larger/smaller cells arising from asymmetric divisions exhibit faster/slower division times than the mean population division time, respectively. On the other hand, cells dividing faster do not do so at the cost of morphological symmetry in division. Finally we show that at suboptimal temperatures the mean distance between the nucleoids increases, explaining the increased variance in division. We conclude that the functionality of E. coli cells is not immune to morphological asymmetries at birth, and that the effectiveness of the mechanism responsible for ensuring the symmetry in division weakens at sub-optimal temperatures.