THE S-LAYER GENE OF LACTOBACILLUS-HELVETICUS CNRZ-892 - CLONING, SEQUENCE AND HETEROLOGOUS EXPRESSION

Lactobacillus helveticus CNRZ 892 contains a surface layer (S-layer) c omposed of protein monomers of 43 kDa organized in regular arrays. The gene encoding this protein (slpH) has been cloned in Escherichia coli and sequenced. slpH consists of 440 codons and is preceded by a ribos ome-binding site (RES) and followed by a putative rho-independent term inator. Indeed, Northern analysis revealed that slpH is a monocistroni c gene. The gene is preceded by a possible promoter of which the -35 a nd -10 hexanucleotides are separated by 17 nt. By primer extension ana lysis the transcription start site was mapped al 7 nt downstream of th e -10 sequence while the deduced amino acid sequence of SlpH shows a l eader peptide of 30 aa. The slpH gene has been amplified by PCR and th e fragment, carrying the complete gene from the RES to the stop codon, has been cloned in a lactococcal gene expression vector downstream of promoter P32. Lactococcus lactis MG1363 carrying the resulting plasmi d produced and secreted an S-layer monomer with the same molecular mas s as the authentic L. helveticus CNRZ 892 SlpH, as judged by SDS-PAGE. Immunoelectron microscopy revealed that SlpH was bound to the lactoco ccal cell walls in small clumps and accumulated in the growth medium a s small sheets.