Final Report Summary - BET-HEDGING BACTERIA (The generality, mechanism, and function of bet-hedging in bacteria) This project has enabled the laboratory to set up multiple projects examining noise in gene expression in bacteria. Several publications are submitted or are near to submission. Work has been carried out to examine how the pulsatile dynamics of alternative sigma factors are modulated by specific stress inducers, and the work has been extended to examine how these sigma factors are expressed in biofilms. The researchers have gone on to build simple mathematical models that simulate the dynamic behaviours that are observed in time-lapse movies. The researchers have also set up off the shelf and custom built microfluidic systems, which have been tested in the laboratory. The laboratory is well set up at the Sainsbury Laboratory, University of Cambridge, with 4 time-lapse microscopes and a group size of 12. The group leader is well established, and has received several other grants, as well as having his position at the Sainsbury Lab renewed until at least 2022. In the future the research will lead to an understanding of how and why alternative states are generated in Bacteria. It is hoped that the work can have long-term implications for public health, by allowing an understanding of how heterogeneous transcriptional states enable bacteria to survive in an ever changing and hostile environment.In collaborative work that has been submitted for publication, the researchers have observed stochastic pulsing behaviour in 7 alternative sigma factors under energy stress. They have also gone onto activate each alternative sigma factor by its specific inducer. Preliminary results suggest a wide range of sigma factor pulse dynamics under stress. For example, sigV shows bimodal activation under lyzosyme stress. They have also constructed a simple mathematical model of sigV activation that suggests that the bimodal activity we observe for sigV under lysozyme stress can be explained by a difference in its regulation compared to sigB where we observe frequency modulated pulsing. In sigV regulation, the anti-sigma factor is targeted for degradation under stress, rather than being sequestered as is the case for the anti-sigma factor of sigB. Simple models can show that this difference in regulation can lead to bi-modality in sigV activity. They are now going to test our predictions from this model. In an addition to the goals set out in the original proposal, the researchers have tested whether heterogeneous activation of sigma factors is observed in bacillus biofilms, in order to check that the variability observed in expression dynamics is not due to the artificial conditions in which cells are grown, in small colonies on agarose pads. B. subtilis grows as a biofilm in the wild, so the researchers wished to check whether heterogeneous expression patterns also occur in biofilms. They have discovered that sigB is expressed heterogeneously in biofilms, and the single cell distributions of sigB activity are qualitatively similar to that observed in liquid culture and in agarose pad movies. They also observe a gradient in sigB expression, with the highest level of activity at the top of the biofilm. They have found interesting competition between the sigB pathway and the sporulation pathway, that the authors are now investigating. They researchers have also successfully setup a microfluidics facility at the Sainsbury Laboratory. They have setup two commercially available microfluidic systems, Cellasic and Celldirector, as well as a custom built Mother Machine variant.