S-layer gene sbsC of Bacillus stearothermophilus ATCC 12980: molecular characterization and heterologous expression in Escherichia coli The GenBank accession number for the sequence described in this paper is AF055578.

Abstract

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 the S-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 115 409 Da and an isoelectric point of 573. 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 the N-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.

Analysis of the structure–function relationship of the S-layer protein SbsC of Bacillus stearothermophilus ATCC 12980 by producing truncated forms

“…Derived from the repeated amino acid sequences within SbsC and from sequence comparison with other S-layer proteins (Jarosch et al, 2000), various N-or C-terminal SbsC truncations (rSbsC…”

“…For cloning, NdeI and BamHI restriction sites were introduced during PCR at the 5h and 3h end, respectively. PCR amplification was performed under conditions given in a previous study (Jarosch et al, 2000). The PCR products encoding the mature SbsC (rSbsC $" -"!…”

“…Typical of protein domains capable of interacting with carbohydrates (Transue et al, 1997 ;Weis, 1997), the N-terminal part of the mature SbsC has a high content of arginine, lysine and tyrosine. In contrast to the mature SbsC, the N-terminal part is positively charged, with a calculated pI of 9n13 (Jarosch et al, 2000). Moreover, the N-terminal part of the mature SbsC shows 85 % identity to the N-terminal region of the mature SbsA, which assembles into a hexagonally ordered lattice and represents the S-layer protein of B. stearothermophilus PV72\p6 .…”

“…The gene encoding this S-layer protein (sbsC) was sequenced and cloned, showing that the full-length SbsC consists of 1099 aa, possesses a 30 aa signal peptide and has a calculated isoelectric point (pI) of 5n73 (Jarosch et al, 2000). In the course of studies on the biological function, the oblique S-layer lattice was found to serve as an adhesion site for a cellassociated high-molecular-mass exoamylase (HMMA) which in the S-layer-deficient strain was bound to the peptidoglycan-containing rigid cell wall layer (Egelseer et al, 1996).…”

A temperature‐sensitive expression system based on the Geobacillus stearothermophilus NRS 2004/3a sgsE surface‐layer gene promoter

“…Studies in various organisms have provided clues to the mechanisms underlying S-layer gene regulation [2]. Frequently, multiple and tandemly arranged promoters are present upstream of S-layer genes [6][7][8][9]. For instance, for the cell-wall protein (cwp) operon of Brevibacillus brevis 47, an organism producing two types of S-layer proteins assembling into two superimposed lattices, three tandemly arranged promoters have been reported.…”