Open AccessSelf‐assembly product formation of the Bacillus stearothermophilus PV72/p6 S‐layer protein SbsA in the course of autolysis of Bacillus subtilis
Author(s) -
Howorka Stefan,
Sára Margit,
Lubitz Werner,
Kuen Beatrix
Publication year - 1999
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1999.tb13468.x
Subject(s) - bacillus subtilis , peptidoglycan , s layer , autolysis (biology) , lysis , cell envelope , cell wall , extracellular , secretion , intracellular , biology , gene product , biophysics , microbiology and biotechnology , signal peptide , biochemistry , chemistry , gene expression , gene , bacteria , escherichia coli , peptide sequence , enzyme , genetics
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.