followed by a putative p-independent terminator. Indeed, Northern analysis
revealed that slpH 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
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 Slayer monomer with the same molecular mass as
the authentic L. helveticus CNRZ 892 SlpH, as judged by SDS-PAGE.
lmmunoelectron microscopy revealed that SlpH was bound to the lactococcal
cell walls in small clumps and accumulated in the growth medium as small
sheets.