Pre-Disposition and Epigenetics Govern Variation in Bacterial Survival upon Stress

<div><p>Bacteria suffer various stresses in their unpredictable environment. In response, clonal populations may exhibit cell-to-cell variation, hypothetically to maximize their survival. The origins, propagation, and consequences of this variability remain poorly understood. Variability persists through cell division events, yet detailed lineage information for individual stress-response phenotypes is scarce. This work combines time-lapse microscopy and microfluidics to uniformly manipulate the environmental changes experienced by clonal bacteria. We quantify the growth rates and RpoH-driven heat-shock responses of individual <em>Escherichia coli</em> within their lineage context, stressed by low streptomycin concentrations. We observe an increased variation in phenotypes, as different as survival from death, that can be traced to asymmetric division events occurring prior to stress induction. Epigenetic inheritance contributes to the propagation of the observed phenotypic variation, resulting in three-fold increase of the RpoH-driven expression autocorrelation time following stress induction. We propose that the increased permeability of streptomycin-stressed cells serves as a positive feedback loop underlying this epigenetic effect. Our results suggest that stochasticity, pre-disposition, and epigenetic effects are at the source of stress-induced variability. Unlike in a bet-hedging strategy, we observe that cells with a higher investment in maintenance, measured as the basal RpoH transcriptional activity prior to antibiotic treatment, are more likely to give rise to stressed, frail progeny.</p> </div

Growth inhibition and clonal cell death correlate with a high stress response.

<p>(A) Correlation between reporter intensity and growth rate in response to stress. The growth rate and fluorescence intensity of single cells are measured 130 minutes after Streptomycin treatment. Red indicates dead cells; blue, live cells. (B) Histogram of the growth rate distribution from the same data set as (A). The dashed line indicates the threshold we chose to distinguish alive from dead cells. (C) Life history of a sub-lineage from the micro-colony (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003148#pgen-1003148-g003" target="_blank">Figure 3</a> for the full lineage tree and the legend therein). The colour code represents the fluorescence intensity. Dead cells are indicated by a red dot at the end of the lineage tree. In addition, the cellular growth rate is represented inversely by line width (e.g., bold line the slow growers). The dashed line indicates the time of induction by streptomycin. Data correspond to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003148#pgen.1003148.s022" target="_blank">Video S2</a>.</p

Increased auto-correlation of the stress response within micro-colonies after induction.

<p>The mathematical derivation of the auto-correlation function (AF) can be found in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003148#pgen.1003148.s020" target="_blank">Text S1</a>. The black curve shows the AF of non-induced micro-colonies (average of 4). The yellow area indicates the standard deviation. The blue, cyan, green and red curves are the AFs calculated with starting points that are 70 minutes, 90 minutes, 110 minutes, and 140 minutes after induction respectively. The induced AF data is from the micro-colony shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003148#pgen.1003148.s022" target="_blank">Video S2</a>. The generation is determined by the number of cells in the micro-colony. All the curves are truncated at the 8–16 cell stage due to increased fluctuations for small sample sizes.</p

Increased cellular membrane permeability following stress induction.

<p>Exponential phase cells of a strain carrying both p<i>^terR</i> - and p<i>^ibpAB</i> -driven fluorescence reporters are plated on LB-agar pads with or without streptomycin (3 µg/ml) and ATC (25 ng/ml). After 2–3 hours of colony growth, the reporter intensity is quantified by fluorescence microscopy. The intensity is normalized to that of the non-induced state ([streptomycin] = [ATC] = 0). The black dot in the middle of each data cloud shows the mean value of both fluorescence channels. R² and k are the coefficient of determination and slope for linear fitting. For each condition, at least 5 micro-colonies are quantified.</p