The S-layer gene of Lactobacillus helveticus CNRZ 892: cloning, sequence and heterologous expression.
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.
Callegari
M Luisa
ML
Istituto di Microbiologia, UCSC, Via Emilia Parmense 84, 29100 Piacenza, Italy.
Riboli
Barbara
B
Istituto di Microbiologia, UCSC, Via Emilia Parmense 84, 29100 Piacenza, Italy.
Sanders
J Willem
JW
Department of Genetics, RUG, Kerklaan 30, 9751NN Haren, The Netherlands.
Cocconcelli
P Sandro
PS
Istituto di Microbiologia, UCSC, Via Emilia Parmense 84, 29100 Piacenza, Italy.
Kok
Jan
J
Department of Genetics, RUG, Kerklaan 30, 9751NN Haren, The Netherlands.
Venema
Gerard
G
Department of Genetics, RUG, Kerklaan 30, 9751NN Haren, The Netherlands.
Morelli
Lorenzo
L
Istituto di Microbiologia, UCSC, Via Emilia Parmense 84, 29100 Piacenza, Italy.
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
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C055764:S-layer proteins