Self-assembly product formation of the Bacillus stearothermophilus PV72/p6 S-layer protein SbsA in the course of autolysis of Bacillus subtilis.
In order to achieve high level expression and to study the release of a protein capable of self-assembly, the gene encoding the crystalline cell surface (S-layer) protein SbsA of Bacillus stearothermophilus PV72/p6, including its signal sequence, was cloned and expressed in Bacillus subtilis. To obtain high level expression, a tightly regulated, xylose-inducible, stably replicating multicopy-plasmid vector was constructed. After induction of expression, the S-layer protein made up about 15% of the total cellular protein content, which was comparable to the SbsA content of B. stearothermophilus PV72/p6 cells. During all growth stages, SbsA was poorly secreted to the ambient cellular environment by B. subtilis. Extraction of whole cells with guanidine hydrochloride showed that in late stationary growth phase cells 65% of the synthesised SbsA was retained in the peptidoglycan-containing layer, indicating that the rigid cell wall layer was a barrier for efficient SbsA secretion. Electron microscopic investigation revealed that SbsA release from the peptidoglycan-containing layer started in the late stationary growth phase at distinct sites at the cell surface leading to the formation of extracellular self-assembly products which did not adhere to the cell wall surface. In addition, intracellular sheet-like SbsA self-assembly products which followed the curvature of the cell became visible in partly lysed cells. Intracellularly formed self-assembly products remained intact even after complete lysis of the rigid cell envelope layer.
Howorka
S
S
Institut für Mikrobiologie and Genetik, Universität Wien, Vienna, Austria.
Sára
M
M
Lubitz
W
W
Kuen
B
B
D001412:Bacillus subtilis / Q000235:genetics* / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002473:Cell Wall / Q000648:ultrastructure; D003593:Cytoplasm / Q000648:ultrastructure; D005822:Genetic Vectors; D001411:Geobacillus stearothermophilus / Q000737:chemistry / Q000235:genetics*; D007150:Immunohistochemistry; D008565:Membrane Proteins; D016253:Microscopy, Immunoelectron; D011994:Recombinant Proteins / Q000096:biosynthesis
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C088619:sbsA protein, Bacillus stearothermophilus