title: 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.
abstract: 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.
authors.affiliation: Center for Ultrastructure Research, Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, 1180 Vienna, Austria. egelseer@edv1.boku.ac.at
authors.forename: E M
authors.initials: EM
authors.lastname: Egelseer
authors.forename: T
authors.initials: T
authors.lastname: Danhorn
authors.forename: M
authors.initials: M
authors.lastname: Pleschberger
authors.forename: C
authors.initials: C
authors.lastname: Hotzy
authors.forename: U B
authors.initials: UB
authors.lastname: Sleytr
authors.forename: M
authors.initials: M
authors.lastname: Sára
mesh_terms: 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
chemical_list: D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D010457:Peptidoglycan; D011108:Polymers; C055764:S-layer proteins