Bacillus subtilis is a soil-inhabiting bacterium, characterized by high-level protein secretion. Since secreted proteins are easy to harvest, B. subtilis is frequently employed in industry for enzyme production. Protein secretion from the cytoplasm, across the cytoplasmic membrane and into the growth medium takes place via the general secretion pathway (Sec) or the so-called twin-arginine (Tat) pathway. Interestingly, B. subtilis cells experience stress when being pushed towards high-level protein secretion. To date, the impact of this secretion stress at the level of the cytoplasmic membrane remained poorly understood, due to the hydrophobic nature and low abundance of membrane proteins. Studies described in this PhD thesis were therefore focused on a definition of protein secretion stress at the membrane level. To this end, a multidisciplinary approach combining biochemical and “omics” technologies was applied. In one series of experiments, the Tat pathway was overexpressed to enhance productivity. This resulted in the identification of a protein that closely interacts with the Tat pathway and modulates its activity. Moreover, high level expression of the Tat pathway resulted in prolonged bacterial growth, altered amino acid metabolism and downregulation of differentiation processes, such as biofilm formation and sporulation. In contrast, absence of the Tat pathway resulted in severe oxidative stress. Lastly, the stressful effects of secreting a staphylococcal antigen via the Sec pathway were analysed by absolute membrane protein quantification, which provided novel insights in the accompanying cellular rearrangements. Altogether, the present studies provide new leads for minimizing secretion stress and enhancing protein production in B. subtilis.
|Kwalificatie||Doctor of Philosophy|
|Datum van toekenning||4-nov-2020|
|Plaats van publicatie||[Groningen]|
|Status||Published - 2020|