title: The S-layer gene of Lactobacillus helveticus CNRZ 892: cloning, sequence and heterologous expression.
abstract: Lactobacillus helveticus CNRZ 892 contains a surface layer (S-layer) composed 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 ribosome-binding site (RBS) and followed by a putative rho-independent terminator. Indeed, Northern analysis revealed that slpH is a monocistronic gene. The gene is preceded by a possible promotor of which the -35 and -10 hexanucleotides are separated by 17 nt. By primer extension analysis the transcription start site was mapped at 7 nt downstream of the -10 sequence while the deduced amino acid sequence of SlpH shows a leader peptide of 30 aa. The slpH gene has been amplified by PCR and the fragment, carrying the complete gene from the RBS to the stop codon, has been cloned in a lactococcal gene expression vector downstream of promoter P32. Lactococcus lactis MG1363 carrying the resulting plasmid produced and secreted an S-layer monomer with the same molecular mass as the authentic L. helveticus CNRZ 892 SlpH, as judged by SDS-PAGE. Immunoelectron microscopy revealed that SlpH was bound to the lactococcal cell walls in small clumps and accumulated in the growth medium as small sheets.
authors.affiliation: Istituto di Microbiologia, UCSC, Via Emilia Parmense 84, 29100 Piacenza, Italy.
authors.forename: M Luisa
authors.initials: ML
authors.lastname: Callegari
authors.affiliation: Istituto di Microbiologia, UCSC, Via Emilia Parmense 84, 29100 Piacenza, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Riboli
authors.affiliation: Department of Genetics, RUG, Kerklaan 30, 9751NN Haren, The Netherlands.
authors.forename: J Willem
authors.initials: JW
authors.lastname: Sanders
authors.affiliation: Istituto di Microbiologia, UCSC, Via Emilia Parmense 84, 29100 Piacenza, Italy.
authors.forename: P Sandro
authors.initials: PS
authors.lastname: Cocconcelli
authors.affiliation: Department of Genetics, RUG, Kerklaan 30, 9751NN Haren, The Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: Kok
authors.affiliation: Department of Genetics, RUG, Kerklaan 30, 9751NN Haren, The Netherlands.
authors.forename: Gerard
authors.initials: G
authors.lastname: Venema
authors.affiliation: Istituto di Microbiologia, UCSC, Via Emilia Parmense 84, 29100 Piacenza, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Morelli
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D015151:Immunoblotting; D007778:Lactobacillus / Q000235:genetics* / Q000378:metabolism; D013294:Lactococcus lactis / Q000235:genetics; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017421:Sequence Analysis; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C055764:S-layer proteins