1887

Abstract

CNRZ 892 contains a surface layer (S-layer) composed of protein monomers of 43 kDa organized in regular arrays. The gene encoding this protein () has been cloned in and sequenced. consists of 440 codons and is preceded by a ribosome-binding site (RBS) and followed by a putative ρ-independent terminator. Indeed, Northern analysis revealed that is a monocistronic gene. The gene is preceded by a possible promoter 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 SIpH shows a leader peptide of 30 aa. The 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. MG1363 carrying the resulting plasmid produced and secreted an S-layer monomer with the same molecular mass as the authentic CNRZ 892 SIpH, as judged by SDS-PAGE. Immunoelectron microscopy revealed that SIpH was bound to the lactococcal cell walls in small clumps and accumulated in the growth medium as small sheets.

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1998-03-01
2021-05-08
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