Temperature-dependence of the single-cell variability in the kinetics of transcription activation in Escherichia coli.

From in vivo single-cell, single-RNA measurements of the activation times and subsequent steady-state active transcription kinetics of a single-copy Lac-ara-1 promoter in Escherichia coli, we characterize the intake kinetics of the inducer (IPTG) from the media, following temperature shifts. For this, for temperature shifts of various degrees, we obtain the distributions of transcription activation times as well as the distributions of intervals between consecutive RNA productions following activation in individual cells. We then propose a novel methodology that makes use of deconvolution techniques to extract the mean and the variability of the distribution of intake times. We find that cells, following shifts to low temperatures, have higher intake times, although, counter-intuitively, the cell-to-cell variability of these times is lower. We validate the results using a new methodology for direct estimation of mean intake times from measurements of activation times at various inducer concentrations. The results confirm that E. coli's inducer intake times from the environment are significantly higher following a shift to a sub-optimal temperature. Finally, we provide evidence that this is likely due to the emergence of additional rate-limiting steps in the intake process at low temperatures, explaining the reduced cell-to-cell variability in intake times.

Increased cytoplasm viscosity hampers aggregate polar segregation in Escherichia coli.

In Escherichia coli, under optimal conditions, protein aggregates associated with cellular aging are excluded from midcell by the nucleoid. We study the functionality of this process under sub-optimal temperatures from population and time lapse images of individual cells and aggregates and nucleoids within. We show that, as temperature decreases, aggregates become homogeneously distributed and uncorrelated with nucleoid size and location. We present evidence that this is due to increased cytoplasm viscosity, which weakens the anisotropy in aggregate displacements at the nucleoid borders that is responsible for their preference for polar localisation. Next, we show that in plasmolysed cells, which have increased cytoplasm viscosity, aggregates are also not preferentially located at the poles. Finally, we show that the inability of cells with increased viscosity to exclude aggregates from midcell results in enhanced aggregate concentration in between the nucleoids in cells close to dividing. This weakens the asymmetries in aggregate numbers between sister cells of subsequent generations required for rejuvenating cell lineages. We conclude that the process of exclusion of protein aggregates from midcell is not immune to stress conditions affecting the cytoplasm viscosity. The findings contribute to our understanding of E. coli's internal organisation and functioning, and its fragility to stressful conditions.

Chromosome and plasmid-borne PLacO3O1 promoters differ in sensitivity to critically low temperatures.

Temperature shifts trigger genome-wide changes in Escherichia coli's gene expression. We studied if chromosome integration impacts on a gene's sensitivity to these shifts, by comparing the single-RNA production kinetics of a PLacO3O1 promoter, when chromosomally-integrated and when single-copy plasmid-borne. At suboptimal temperatures their induction range, fold change, and response to decreasing temperatures are similar. At critically low temperatures, the chromosome-integrated promoter becomes weaker and noisier. Dissection of its initiation kinetics reveals longer lasting states preceding open complex formation, suggesting enhanced supercoiling buildup. Measurements with Gyrase and Topoisomerase I inhibitors suggest hindrance to escape supercoiling buildup at low temperatures. Consistently, similar phenomena occur in energy-depleted cells by DNP at 30 °C. Transient, critically-low temperatures have no long-term consequences, as raising temperature quickly restores transcription rates. We conclude that the chromosomally-integrated PLacO3O1 has higher sensitivity to low temperatures, due to longer-lasting super-coiled states. A lesser active, chromosome-integrated native lac is shown to be insensitive to Gyrase overexpression, even at critically low temperatures, indicating that the rate of escaping positive supercoiling buildup is temperature and transcription rate dependent. A genome-wide analysis supports this, since cold-shock genes exhibit atypical supercoiling-sensitivities. This phenomenon might partially explain the temperature-sensitivity of some transcriptional programs of E. coli.

Single-cell kinetics of a repressilator when implemented in a single-copy plasmid.

Synthetic genetic clocks, such as the Elowitz-Leibler repressilator, will be key regulatory components of future synthetic circuits. We constructed a single-copy repressilator (SCR) by implementing the original repressilator circuit on a single-copy F-plasmid. After verifying its functionality, we studied its behaviour as a function of temperature and compared it with that of the original low-copy-number repressilator (LCR). Namely, we compared the period of oscillations, functionality (the fraction of cells exhibiting oscillations) and robustness to internal fluctuations (the fraction of expected oscillations that would occur). We found that, under optimal temperature conditions, the dynamics of the two systems differs significantly, although qualitatively they respond similarly to temperature changes. Exception to this is in the functionality, in which the SCR is higher at lower temperatures but lower at higher temperatures. Next, by adding IPTG to the medium at low and high concentrations during microscopy sessions, we showed that the functionality of the SCR is more robust to external perturbations, which indicates that the oscillatory behaviour of the LCR can be disrupted by affecting only a few of the copies in a cell. We conclude that the SCR, the first functional, synthetic, single-copy, ring-type genetic clock, is more robust to lower temperatures and to external perturbations than the original LCR. The SCR will be of use in future synthetic circuits, since it complements the array of tasks that the LCR can perform.