Self-assembly product formation of the Bacillus stearothermophilus PV72/p6S-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 pro tein capable of self-assembly, the gene encoding the crystalline cell surfa ce (S-layer) protein SbsA of Bacillus stearothermophilus PV72/p6, including its signal sequence, was cloned and expressed in Bacillus subtilis. To obt ain high level expression, a tightly regulated, xylose-inducible, stably re plicating multicopy-plasmid vector was constructed. After induction of expr ession, 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 a mbient cellular environment by B. subtilis. Extraction of whole cells with guanidine hydrochloride showed that in late stationary growth phase cells 6 5% of the synthesised SbsA was retained in the peptidoglycan-containing lay er, indicating that the rigid cell wall layer was a barrier for efficient S bsA secretion. Electron microscopic investigation revealed that SbsA releas e from the peptidoglycan-containing layer started in the late stationary gr owth phase at distinct sites at the cell surface leading to the formation o f extracellular self-assembly products which did not adhere to the cell wal l surface. In addition, intracellular sheet-like SbsA self-assembly product s which followed the curvature of the cell became visible in partly lysed c ells. Intracellularly formed self-assembly products remained intact even af ter complete lysis of the rigid cell envelope layer. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.