S-layer gene sbsC of Bacillus stearothermophilus ATCC 12980: molecular characterization and heterologous expression in Escherichia coli

Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur, 1180 Vienna, Austria 1 Institut für Angewandte Mikrobiologie, Universität für Bodenkultur, 1190 Vienna, Austria 2 Author for correspondence: Margit Sára. Tel: +43 1 47 654 2208. Fax: +43 1 47 89112. e-mail: sara{at}edv1.boku.ac.at The cell surface of Bacillus stearothermophilus ATCC 12980 is completely covered with an oblique S-layer lattice. To investigate sequence identities and a common structure–function relationship in S-layer proteins of different B. stearothermophilus wild-type strains, the nucleotide sequence encoding theS-layer protein SbsC of B . stearothermophilus ATCC 12980 was determined by PCR techniques. The entire sbsC sequence showed an ORF of 3297 bp predicted to encode a protein of 1099 aa with a theoretical molecular mass of 115409 Da and an isoelectric point of 5·73. Primer extension analysis suggested the existence of two promoter regions. Amino acid sequence comparison between SbsC and SbsA, a previously characterized S-layer protein of B. stearothermophilus PV72/p6 which assembles into a hexagonally ordered lattice, revealed an identical secretion signal peptide, 85% identity for theN-terminal regions (aa 31–270) which do not carry any S-layer homologous motifs, but only 21% identity for the rest of the sequences. Affinity studies demonstrated that the N-terminal part of SbsC is necessary for recognition of a secondary cell wall polymer. This was in accordance with results obtained in a previous study for SbsA, thus confirming a common functional principle for the N-terminal parts of both S-layer proteins. The sbsC coding region cloned into the pET3a vector without its own upstream region, the signal sequence and the 3' transcriptional terminator led to stable expression in Escherichia coli . Keywords: S-layer protein, Bacillus stearothermophilus , promoter, heterologous expression, secondary cell wall polymer Abbreviations: SCWP, secondary cell wall polymer; SLH, S-layer homologous The GenBank accession number for the sequence described in this paper is AF055578 .