Secretion processes as a limiting factor of protein production in Bacillus

Summary Protein secretion involves several important sequential steps. First, proteins to be secreted must be recognized and their translocation-competent conformation must be ensured. This is followed by the overcoming of two barriers, the cell membrane and the cell wall. The active transport across the membrane can occur by several well-studied mechanisms, the most notably of them are known as "general secretory" (Sec) and "twin-arginine translocation" (Tat). For the passage through the cell wall, on the other hand, understanding is still almost completely lacking. In this work, I investigated this process, using super-resolution fluorescence microscopy to visualize AmyE-mCherry during secretion in Bacillus subtilis and Bacillus licheniformis. The overexpressed fusion protein localized as distinct foci in the cell envelope, which were mostly lost upon degradation of the bacterial cell wall through treatment with lysozyme. I could also show that AmyE is released from the cells at discrete zones, similar to the localization of fluorescently labeled AmyE as foci inside the envelope. High-level protein secretion peaked at the transition from exponential growth to the stationary phase and appears to be restricted to a subpopulation of cells, which presumably is also the case for general protein secretion. Time lapse experiments revealed the AmyE-mCherry foci to be statically positioned throughout several minutes, in contrast to the lateral mobility of Secmachinery associated membrane proteins SecA and SecDF, labeled with mNeonGreen. Interestingly, the AmyE-mCherry foci displayed considerable fluctuations of fluorescence intensities within a minutes-time-scale, suggesting visualized diffusion of proteins along the passage through the cell walls meshwork. This idea of diffusion is supported by recent AFM Imaging results of B. subtilis, revealing a heterologous cell wall structure with deep pores its peptidoglycan surface. For large parts of industrial biotechnology, the secretion of microbially produced enzymes and ...