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 amino acids long, called S-layer homology (SLH) motifs. Bacillus anthracis, the causal agent of anthrax, synthesizes two S-layer proteins, each with three SLH motifs towards the amino-terminus. We used biochemical and genetic approaches to investigate the involvement of these motifs in cell surface anchoring. Proteinase K digestion produced polypeptides lacking these motifs, and stable three-motif polypeptides were produced in Escherichia coli 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 chimeric genes encoding the SLH domains fused to the normally secreted levansucrase of Bacillus subtilis. Cell fractionation and electron microscopy studies showed that each three-motif domain was sufficient for the efficient anchoring of levansucrase onto the cell surface. Proteins consisting of truncated SLH domains fused to levansucrase were unstable and associated poorly with the cell surface. Surface-exposed levansucrase retained its enzymatic and antigenic properties.
Mesnage
S
S
Toxines et Pathogénie Bactériennes (URA 1858, CNRS), Paris, France. smesnage@pasteur.fr
Tosi-Couture
E
E
Fouet
A
A
D000595:Amino Acid Sequence; D001408:Bacillus anthracis / Q000737:chemistry* / Q000201:enzymology; D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002473:Cell Wall / Q000378:metabolism; D019286:Endopeptidase K / Q000494:pharmacology; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins; D006602:Hexosyltransferases; C021513:levansucrase; D019286:Endopeptidase K