Production and cell surface anchoring of functional fusions between the SLH motifs of the Bacillus anthracis S-layer proteins and the Bacillus subtilis levansucrase

Many surface proteins of Gram-positive bacteria contain motifs, about 50 am ino acids long, called S-layer homology (SLH) motifs, Bacillus anthracis, t he causal agent of anthrax, synthesizes two S-layer proteins, each with thr ee SLH motifs towards the amino-terminus, We used biochemical and genetic a pproaches to investigate the involvement of these motifs in cell surface an choring. Proteinase K digestion produced polypeptides lacking these motifs, and stable three-motif polypeptides were produced in Escherichia coil that were able to bind the B. anthracis cell walls in vitro, demonstrating that the three SLH motifs were organized into a cell surface anchoring domain. We also determined the function of these SLH domains by constructing chimer ic genes encoding the SLH domains fused to the normally secreted levansucra se of Bacillus subtilis. Cell fractionation and electron microscopy studies showed that each three-motif domain was sufficient for the efficient ancho ring of levansucrase onto the cell surface. Proteins consisting of truncate d SLH domains fused to levansucrase were unstable and associated poorly wit h the cell surface. Surface-exposed levansucrase retained its enzymatic and antigenic properties.