Two types of transcriptional repression in living cells of [i]Bacillus subtilis[/i] characterized by number and brightness analysis

Number and brightness analysis (N&B) is a useful technique for characterizing the concentration and molecular brightness of fluorescent molecules in vivo. Here we investigate stochastic gene expression in Catabolite Repression in Bacillus subtilis. In particular the transcriptional activity of promoters implicated in the switch between glycolysis and gluconeogenesis was investigated. Promoter activity was measured using green fluorescent protein (GFP) promoter fusions. Two photon laser scanning microscopy and true N&B analysis allowed determination of the absolute concentration of GFP molecules inside the bacterial cells. We collected data on hundreds of B. subtilis cells expressing GFP under control of the promoters of interest and grown under glycolytic or gluconeogenic conditions. Results showed no regulation of the promoter expressing the gluconeogenic repressor, strong repression of the gluconeogenic enzyme promoters and weak auto-repression of the glycolytic promoter, with a highly asymmetric distribution when repressed. All promoters showed strong evidence for transcriptional bursting. Analysis of the data using stochastic models of gene expression is currently underway. The figure shows number maps of bacterial cells grown on glucose(G) or Malate(M). Each change in color represents 10 molecules up to 180.View Large Image | View Hi-Res Image | Download PowerPoint Slide