We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics.

We studied whether nucleoid exclusion contributes to the segregation and retention of Tsr chemoreceptor clusters at the cell poles. Using live time-lapse, single-cell microscopy measurements, we show that the single-cell spatial distributions of Tsr clusters have heterogeneities and asymmetries that are consistent with nucleoid exclusion and cannot be explained by the diffusion-and-capture mechanism supported by Tol-Pal complexes at the poles. Also, in cells subjected to ampicillin, which enhances relative nucleoid lengths, Tsr clusters locate relatively closer to the cell extremities, whereas in anucleated cells (deletion mutants for mukB), the Tsr clusters are closer to midcell. In addition, we find that the fraction of Tsr clusters at the poles is smaller in deletion mutants for Tol-Pal than in wild-type cells, although it is still larger than would be expected by chance. Also in deletion mutants, the distribution of Tsr clusters differs widely between cells with relatively small and large nucleoids, in a manner consistent with nucleoid exclusion from midcell. This comparison further showed that diffusion-and-capture by Tol-Pal complexes and nucleoid exclusion from the midcell have complementary effects. Subsequently, we subjected deletion mutants to suboptimal temperatures that are known to enhance cytoplasm viscosity, which hampers nucleoid exclusion effects. As the temperature was lowered, the fraction of clusters at the poles decreased linearly. Finally, a stochastic model including nucleoid exclusion at midcell and diffusion-and-capture due to Tol-Pal at the poles is shown to exhibit a cluster dynamics that is consistent with the empirical data. We conclude that nucleoid exclusion also contributes to the preference of Tsr clusters for polar localization.

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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.

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.

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