Characterization of an S-layer glycoprotein produced in the course of S-layer variation of Bacillus stearothermophilus ATCC 12980 and sequencing and cloning of the sbsD gene encoding the protein moiety.
The cell surface of Bacillus stearothermophilus ATCC 12980 is completely covered by an oblique lattice which consists of the S-layer protein SbsC. On SDS-polyacrylamide gels, the mature S-layer protein migrates as a single band with an apparent molecular mass of 122 kDa. During cultivation of B. stearothermophilus ATCC 12980 at 67 degrees C instead of 55 degrees C, a variant developed that had a secondary cell wall polymer identical to that of the wild-type strain, but it carried an S-layer glycoprotein that could be separated on SDS-polyacrylamide gels into four bands with apparent molecular masses of 92, 118, 150 and 175 kDa. After deglycosylation, only a single protein band with a molecular mass of 92 kDa remained. The complete nucleotide sequence encoding the protein moiety of this S-layer glycoprotein, termed SbsD, was established by PCR and inverse PCR. The sbsD gene of 2,709 bp is predicted to encode a protein of 96.2 kDa with a 30-amino-acid signal peptide. Within the 807 bp encoding the signal peptide and the N-terminal sequence (amino acids 31-269), different nucleotides for sbsD and sbsC were observed in 46 positions, but 70% of these mutations were silent, thus leading to a level of identity of 95% for the N-terminal parts. The level of identity of the remaining parts of SbsD and SbsC was below 10%, indicating that the lysine-, tyrosine- and arginine-rich N-terminal region in combination with a distinct type of secondary cell wall polymer remained conserved upon S-layer variation. The sbsD sequence encoding the mature S-layer protein cloned into the pET28a vector led to stable expression in Escherichia coli HMS174(DE3). This is the first example demonstrating that S-layer variation leads to the synthesis of an S-layer glycoprotein.
Egelseer
E M
EM
Center for Ultrastructure Research, Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, 1180 Vienna, Austria. egelseer@edv1.boku.ac.at
Danhorn
T
T
Pleschberger
M
M
Hotzy
C
C
Sleytr
U B
UB
Sára
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism*; D014644:Genetic Variation; D001411:Geobacillus stearothermophilus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010457:Peptidoglycan / Q000032:analysis; D011108:Polymers / Q000032:analysis; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D010457:Peptidoglycan; D011108:Polymers; C055764:S-layer proteins