Rapid Transcription Factor Fluctuations Drive Nonequilibrium Gene Regulatory Dynamics in Bacteria

In bacteria, fluctuations in transcription factor (TF) concentrations and stochastic binding to promoters drive gene expression fluctuations in their target genes. However, how this “noise propagation” from TFs to their targets determines the expression fluctuations in target genes has so far not been quantitatively characterized. Here, we use fluorescence time-lapse microscopy in combination with microfluidics and automated image analysis to quantitatively track the single-cell expression dynamics of target promoters of the SOS response regulator LexA in under mild DNA damage using the antibiotic Ciprofloxacin (Cipro). We find that all target promoters exhibit short stochastic expression bursts which become more frequent as the Cipro concentration increases, but whose durations are identical for all promoters, with exponentially distributed heights that vary across promoters, but that are independent of Cipro concentration. We show that these observations are inconsistent with popular “equilibrium” models of bacterial gene regulation that assume that binding and unbinding of TFs to promoters is fast relative to TF concentration fluctuations. Instead, all observations are quantitatively fit by a model in which DNA damage events lead to rapid transient dips in LexA concentration, and an expression burst occurs when LexA unbinds from the target promoter during such a short dip. Crucially, because these LexA concentration fluctuations occur on the same timescale as individual TF unbinding events, the responses of different target promoters to individual DNA damage events are complex stochastic and nonequilibrium functions of the strengths of their LexA binding sites and the durations of the events. Our results show that when induction events cause rapid TF concentration changes, i.e., on the timescale of individual TF binding and unbinding events, different target promoters exhibit distinct stochastic responses that read out different kinetics of the induction events. Since many bacterial TFs are activated through phosphorylation or binding to small signaling molecules, such nonequilibrium gene regulation is likely pervasive in bacteria, and may have been exploited by natural selection to encode distinct responses by different targets of the same TF.