CLONING OF A GENE ENCODING NISIN RESISTANCE FROM LACTOCOCCUS-LACTIS SUBSP LACTIS M189 WHICH IS TRANSCRIBED FROM AN EXTENDED -10-PROMOTER

A 56-kb plasmid was identified in Lactococcus lactis subsp. lactis (L. lactis) M189 which encodes resistance to nisin (Nis(R)) following mob ilization of the plasmid into L. lactis LM0230. The Nis(R) determinant was localized on a 1.6-kb HindIII fragment by DNA restriction fragmen t deletion and subcloning. An open reading frame (ORF) of 957 bases wa s identified by sequence analysis and its transcription start site was mapped by primer extension. The ORF is flanked by two regions which e xhibit complete homology to parts of the inverted repeat sequences of IS981 and ISS1T. The promoter for transcription was found to consist o f an extended -10 site (TgTGtTATAAT) that lacks a -35 site. Function o f the extended -10 promoter was demonstrated by its ability to express the promoterless cat gene from Staphylococcus aureus. Base substituti on analysis revealed that the TgTGt extension is essential for promote r efficiency in L. lactis.