S-layer protein gene of Lactobacillus brevis: cloning by polymerase chain reaction and determination of the nucleotide sequence.
The surface (S)-layer protein of Lactobacillus brevis was isolated, purified, and characterized. The S-layer protein is the major protein of the cell, with an apparent molecular mass of 46 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunogold electron microscopy with polyclonal antiserum against the isolated 46-kDa protein was used to confirm the surface location of this protein. N-terminal amino acid sequences of the intact 46-kDa protein and its tryptic peptides were determined. The gene of the S-layer protein was amplified from the genome of L. brevis by polymerase chain reaction with oligonucleotides, synthesized according to the N-terminal amino acid sequences, as primers. The polymerase chain reaction fragments containing the entire S-layer gene and its regulatory regions were sequenced. Nucleic acid sequence analysis revealed one open reading frame with a capacity to encode a protein of 48,159 Da. From the regulatory region of the gene, two subsequent promoters and a ribosome binding site, showing typical features of prokaryotic consensus sequences, were found. The coding region contained a characteristic gram-positive-type signal peptide of 30 amino acids. Removal of the signal peptide results in a polypeptide of 435 amino acids, which is in excellent agreement with the size of the S-layer protein determined by SDS-PAGE. The size and the 5' end analyses of the S-layer transcripts confirmed the monocistronic nature of the S-layer operon and the functionality of the two promoters found.
Vidgrén
G
G
Institute of Biotechnology, University of Helsinki, Finland.
Palva
I
I
Pakkanen
R
R
Lounatmaa
K
K
Palva
A
A
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000235:genetics* / Q000648:ultrastructure; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003062:Codon; D004269:DNA, Bacterial; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D012333:RNA, Messenger; C055764:S-layer proteins