ETD

• acid adaptation
• acid stress
• Escherichia coli
• fluorescence activated cell sorting
• green fluorescent protein
• O157
• pathogenic strains of Escherichia coli

Many microorganisms have a remarkable adaptability at pH values significantly or relatively lower compared with the optimal values of growth.
Between these microorganisms it is possible to include Escherichia coli, which has developed four systems response to acid stress. The two best-studied systems are characterized by the presence of amino acid-dependant decarboxylase: isozymes GadA and GadB, glutamate dependant, belonging to acid stress response system 2, and the decarboxylase AdiA, employee from arginine, and present in the system response 3. The response to stress is governed by a dense network of genes that cooperate with each other through an activation cascade of the promoters. The results of transcriptomic analysis from microaarays made it possible to determine which are the promoters triggered by lower pH. The following step in our experiments was the selection of some of the promoters who have shown this resistance and subsequent analysis by Fluorescence Activated Cell Sorting, assessing the fluorescence of the Green Fluorescence Protein inserted in reporter constructs. Experiments were done on a library of constructs containing the promoters of E. Coli K-12. The assessment of fluorescence was done in the same conditions that occur in a chemostat, using shake flasks, therefore trying to maintain bacterial growth steady and stable through continuous replenishment of the media culture after the acidification.
FACS is an investigation method to measure transcriptional activity at the population level, which cannot be done with other transcriptional readouts. The use of this technique in these experiments is just a way to optimize this scientific approach and to obtain even more results and reproducibility with many promoters of the GFP library, trying to understand which are the genes involved in acid stress response that can belong to important metabolic pathways. Future works will be directed towards developing a dataset with a large number of time points and by applying bioiformatics tools, attempting to formulate hypothesis on the regulatory networks underlying this tress response.