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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 ac...
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| Format: | Doctoral Thesis |
| Language: | English |
| Published: |
Philipps-Universität Marburg
2023
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| Online Access: | PDF Full Text |
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| Summary: | 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
proteins into the culture supernatant is of enormous relevance, due to the lower costs for subsequent
processing associated with this method as compared to the disruption of the producing cells. Studies
investigating secretion efficiency in Bacillus species, have revealed numerous influencing factors.
Since the bacterial cell wall is often overlooked in the search for secretion bottlenecks, I targeted
autolysins that can affect cell wall thickness and the density of the meshwork. While absence of
LytD had little effect on secretion, deletion of lytC and lytF significantly impaired AmyE transport
to the outside of the cell. By introducing additional genes encoding the autolysins LytC and LytF or
the cell wall hydrolase PBP5 (dacA), I was able to improve secretion by up to 200%. These findings
suggest that cell wall permeability for secreted proteins is modulated by autolysin activity.
Flotillins, which are thought to form functional membrane microdomains (FMM) in B. subtilis, are
often linked with secretion, although the nature of this connection is not exactly clear. To approach
this subject, I used a ΔyuaG (FloT) deletion strain with reduced AmyE secretion and showed that
the addition of the membrane fluidizer benzyl alcohol could recover the AmyE secretion level of the
wild type. This result indicates, that flotillins affect protein secretion in B. subtilis through the ability
to improve membrane fluidity. Furthermore, I was able to double the efficiency of AmyE secretion
of B. subtilis by introducing an additional gene encoding FloT. |
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| DOI: | 10.17192/z2023.0677 |