PhD ceremony Mr. S. Piersma: Applications of quantitative fluorescence microscopy for systems level gene expression analyses in Bacillus subtilis

PhD ceremony: Mr. S. Piersma

Dissertation: Applications of quantitative fluorescence microscopy for systems level gene expression analyses in Bacillus subtilis

Promotor(s): prof. J.M. van Dijl

Faculty: Medical Sciences

The present PhD research was aimed at the development of novel tools for quantitative fluorescence microscopy. These were needed for systems-level gene expression analyses in the soil bacterium Bacillus subtilis, a popular model organism for research on bacterial gene expression. The studies were rooted in the EC-funded BaSysBio project and started with the construction of an arrayed collection of more than 1000 different fusions between promoters of B. subtilis and the Green Fluorescent Protein (GFP). Each GFP fusion was expressed in a different strain and, together, this strain collection formed a so-called Live Cell Array (LCA). The LCA was then employed in genome-wide gene expression analyses in response to changing nutritional conditions. These analyses were performed at the population level in microtiter plate readers, and at the single-cell level using high-throughput time-lapse fluorescence microscopy. For the latter purpose, a dedicated data processing pipeline (TLM-Quant) was developed based on open-source software. The BaSysBio project culminated in a publication in Science describing the regulatory network of B. subtilis at the transcriptome, proteome and metabolome levels. Here, the LCA and TLM-Quant were employed to verify gene expression homogeneity in exponentially growing cells. Subsequently, the developed tools were applied in several other investigations on the origin of heterogeneous expression of an antimicrobial agent, the bacterial responses to limited water availability, the secretion of proteins, and bacterial responses to a novel antimicrobial compound. Altogether, this PhD research expanded the toolbox for quantitative microscopy especially through the development of methods for faster data processing and visualization.