Identification of a region responsible for binding to the cell wall within the S-layer protein of Clostridium thermocellum

Abstract

The protomer forming the Slayer of CIostridium thermocellum was identified as a 140 kDa protein which was non-covalently bound to the cell wall. Cloning and sequencing of the corresponding gene revealed an open reading frame of 3108 nucleotides encoding a polypeptide of 1036 amino acids, termed SlpA. The amino acid composition of SlpA matches the composition of a previously described exocellular glycoprotein. SlpA shared extensive similarity with the Slayer protein of Bacillus sphaericus and with the outer wall protein of Bacillus brewis. In addition, the amino-terminal region of SlpA contained a segment presenting similarities with segments termed SLH (Slayer homologous), which are found in several bacterial exoproteins. A polypeptide of 209 residues comprising this segment was shown to bind to cell walls extracted from C. thermoeellum cells.

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

“…a et al, Truncated forms of the S-layer protein SbsC 1996). In contrast, the N-terminal parts of the S-layer proteins Sap and EA1 of Bacillus anthracis (Mesnage et al, 1999) and SlpA of Clostridium thermocellum (Lemaire et al, 1998 ;Chauvaux et al, 1999) comprising the three SLH motifs were able to attach to the rigid cell wall layer via a peptidoglycan-associated cell wall polymer. As demonstrated in previous studies, a proteolytic cleavage fragment of the S-layer protein SbsC from B. stearothermophilus ATCC 12980 lacking the N-terminal 227 aa had lost the ability to bind to the native peptidoglycan-anionic polymer complex, but was still able to self-assemble Jarosch et al, 2000).…”

A general strategy for identification of S-layer genes in the Bacillus cereus group: molecular characterization of such a gene in Bacillus thuringiensis subsp. galleriae NRRL 4045 The GenBank accession number for the slpA sequence is AJ249446.

“…1, with BT23 and BT24) (Trieu-Cuot et al, 1991). It is not advisable to clone the gene with its regulatory region as this could lead to problems and in many cases has proved impossible (Lemaire et al, 1998 and references therein). The gene is then disrupted by insertion of a selectable marker, such as the spectinomycin resistance cassette inserted into the SapI site in this study, and the copy harbouring the resistance cassette is integrated into the Bac.…”

S-Layer Proteins

“…Due to their wide distribution among gram-positive bacteria, SLH motifs were suggested to anchor cell-associated exoproteins permanently or transiently to the cell surface (74). In contrast to the original assumption that peptidoglycan functions as a binding site (74), it is now evident that secondary cell wall polymers serve as anchoring structures for many S-layer proteins, cellassociated exoenzymes, and exoproteins (15,22,51,72,73,83,107).…”

“…By sequence comparison, S-layer-homologous (SLH) motifs (74) have been identified at the N-terminal part of many Slayer proteins (14,26,33,39,40,51,65,71,72,85,97,144) and at the C-terminal end of cell-associated exoenzymes (71,78,79) and other exoproteins (71, 73) of gram-positive bacteria. According to their origin (S-layer proteins, cell-associated exoproteins, porins), SLH motifs have been divided into three main groups whose specific properties have recently been reviewed by Engelhardt and Peters (38).…”