Regulation of immunity to the two-component lantibiotic, lacticin 3147, bythe transcriptional repressor LtnR

Lacticin 3147 is a membrane-active, two-component lantibiotic produced by L actococcus lactis ssp. lactis DPC3147. In this study, the promoters of the lacticin 3147 gene cluster were mapped to the intergenic region between Itn R and ItnA(1) (the genes encoding the regulatory protein LtnR and the first structural gene, LtnA1), and Northern analyses revealed that the biosynthe tic and immunity genes are divergently transcribed in two operons, ItnA(1)A (2)M(1)TM(2)D and ItnRIFE respectively. Although the promoter controlling b iosynthesis (P-bac) appears to be constitutive, characterization of a downs tream beta -galactosidase (beta -gal) fusion beyond an intragenic stem-loop structure in ItnM(1) confirmed that this putative transcriptional attenuat or allows limited readthrough to the downstream biosynthetic genes, thus ma intaining the correct stoichiometry between structural peptides and biosynt hetic machinery. The promoter of the ItnRIFE operon (P-imm) was shown to be regulated by the transcriptional repressor LtnR. A mutant with a truncated ItnR gene exhibited a hyperimmune phenotype, whereas overexpression of Itn R resulted in cells with increased sensitivity to lacticin 3147. Gel mobili ty shift analysis indicated that LtnR binds to the P-imm promoter region, a nd fusion of this promoter to the beta -gal gene of pAK80 revealed that exp ression from P-imm is significantly reduced in the presence of LtnR. Thus, we have demonstrated that lacticin 3147 uses a regulatory mechanism not pre viously identified in lantibiotic systems.