title: Dynamics of cell wall binding proteins and membrane-associated secretion proteins at a single molecule level
Creators: name: Fiedler, Svenja Mareike
Publisher: name: Philipps-Universität Marburg
Year: 2023
Subjects: dynamics
Sec-system
FtsY
B. subtilis
hydrolases
SRP
Natural sciences &amp; mathematics
Naturwissenschaften
Cell wall
SecA
autolysins
SecYEG
SMT

description: Protein secretion is an essential mechanism for cells, which has already been well-researched
in Gram-negative bacteria. In Gram-positive bacteria, however, homologs of known proteins
of the mechanism are missing, and how these are compensated is still unclear. A key factor in
protein secretion is SecA, which acts as a motor protein that pushes secretory proteins through
the SecYEG translocon. In Gram-negative bacteria, this protein is supported by the holdase
SecB. The interaction between SecA and SecB is not essential but increases efficiency.
In this work, several proteins involved in the secretion process were analyzed by single
molecule tracking. Unlike in E. coli, SecA of B. subtilis is not mainly located at the membrane,
where it binds to the Sec-translocon, but is predominantly found in the cytoplasm. The SecA
molecules are localized primarily in areas outside the nucleoid and copy the localization of
ribosomes, suggesting that SecA picks up secretory proteins directly at the ribosome and that
SecA in B. subtilis can take over the tasks of SecA and SecB in E. coli. It was also shown that
the size of the static population, which represents SecA engaged in substrate translocation,
depends on the growth phase of the cell culture, indicating adaptation of SecA dynamics
according to the distinct cellular requirements.
Furthermore, the behavior of SecA, SecDF, YidC, and FtsY was investigated in case of an
overproduction of the secretory protein AmyE. It was found that not only SecA becomes more
strongly engaged in substrate translocation during overproduction, but also FtsY, belonging to
the signal recognition particle pathway for integration of membrane proteins. Proteins SecDF,
implicated in secretion, and YidC, a membrane integrase, do not change their single molecule
behavior. These data suggest that the SRP system might take over SecA substrate proteins
during overexpression conditions.
A large class of secretory proteins translocated by the SecA system are cell wall hydrolases
(autolysins). To better understand how they move within and/or along the cell wall, the
dynamics of LytC and LytF were analyzed. LytC is one of the main autolysins in B. subtilis and
localizes around the cell, whereas LytF is mainly involved in cell division and is, therefore,
mostly found at the septa and poles. A three-population fit was found to be the best for both
proteins. The fastest diffusion constant was comparable to freely diffusing proteins observed
in the cytoplasm, which would support the existence of a periplasmic space. Molecules
belonging to the population with a slow mobile diffusion constant are most likely proteins that
move along the cell wall searching for targets. For comparison, the observed diffusion constant
is similar to that of DNA-binding proteins sliding through the genome. The static population,
confined to the periphery of cells, likely corresponds to autolysins engaged in substrate
hydrolysis. The investigation of the dynamics of the cell wall hydrolases LytC and LytF inV
different deletion strains showed that the localization of the proteins is not only determined by
the cell wall binding domains, as stated in the literature, but also by other cell wall hydrolases
and inhibitory proteins, whose absence strongly influenced the dynamics as well as preferred
dwell sites of the investigated cell wall hydrolases. These findings indicate an intricate interplay
of the many redundant autolysins and their inhibitors within the cell wall.
With regard to secretion efficiency, this work revealed a possible connection between cell wall
hydrolases and protein secretion, indicating that the activity of cell wall hydrolases increases
the secretion rate of AmyE. This finding supports the idea that the secretion of large proteins
through the cell wall requires areas of loosened connections in the cell wall for the passage of
proteins into the surrounding environment.