Bistable differentiation in an isogenic cell population

Abstract

Single-cell organisms such as bacteria have traditionally been regarded as discrete units, which in turn has been reflected by the bulk-level methods used to study them. A growing culture of the bacterium Bacillus subtilis will exhibit a range of heterogeneous genetic developmental programmes such as motility, competence, and finally sporulation. As a popular choice for production of compounds in bioreactors, the bistable behaviours of B. subtilis may be undesirable traits, as they divert resources from their intended activity of synthesising a product. This thesis investigates a novel observation that expression of a ribosomal subunit gene (rpsD) is elevated in the non-motile state of B. subtilis, using unstable GFP reporter constructs. The implications of using a proteolytically unstable protein as a reporter are also investigated with regard to the effect of protein degradation rates on the reporter construct, as well as presenting evidence for modulation of ClpXP activity in a pnpA background. Investigation of the motile/non-motile heterogeneous phenotype of B. subtilis posed a challenge for automated analysis pipelines. This thesis addresses this problem by developing and testing microscopy analysis pipelines designed to circumvent the traditional requirement for physically separated objects in a phase contrast channel, and instead using nucleoid or membrane stains to identify cells in a microscopy image. Other factors impacting the activity of a proteolytically unstable PrpsD reporter construct were investigated, including the rate of degradation of the reporter, and integration locus of the reporter construct. To assess the impact of locus positioning, a genetic tool was also created to survey changes in noise and overall expression levels from two homogeneously expressed promoters across different positions on the chromosome.