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

Despite its possible role in virulence, there has been little molecular cha racterization of members of the S-layer protein family of the Bacillus cere us group. It is hypothesized that the components of the S-layers are likely to display similar anchoring structures in Bacillus thuringiensis and Baci llus anthracis. Based on inferred sequence similarities, a DNA fragment enc oding the cell-wall-anchoring domain of an S-layer component of Bac. thurin giensis subsp. galleriae NRRL 4045 was isolated. The complete gene was iden tified and sequenced. An ORF of 2463 nt was identified, which was predicted to encode a protein of 821 aa, SlpA. The mature SlpA protein (792 residues ) carries three S-layer homology (SLH) motifs towards its amino terminus, e ach about 50 aa long. These motifs were sufficient to bind Bac. thuringiens is and Bac. anthracis cell walls in vitro by interacting with peptidoglycan -associated polymers, confirming a common wall-anchoring mechanism. The slp A null-allele mutant was constructed and shown to possess no other abundant surface protein. It is proposed that the method described in this paper co uld be applied to the identification and deletion of any Bac. cereus S-laye r gene and is of great value for the molecular and functional characterizat ion of these genes.