Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105 M.C. Martõ Ânez-Cuesta 1 , G. Buist 2 , J. Kok 2 , H.H. Hauge 3 , J. Nissen-Meyer 3 , C. Pela  ez 1 and T. Requena 1 1 Department of Dairy Science and Technology, Instituto del Frõ Âo, Ciudad Universitaria, Madrid, Spain, 2 Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen,The Netherlands, and 3 Department of Biochemistry, University of Oslo, Norway 111/12/99: received 23 December 1999, revised 20 March 2000 and accepted 22 March 2000 M.C. MARTI  NEZ-CUESTA, G. BUIST, J. KOK, H.H. HAUGE, J. NISSEN-MEYER, C. PELA  EZ AND T. REQUENA. 2000. The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was puri®ed to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (a and b) with average molecular masses of 3322 and 2848 Da, respectively. The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L. lactis DPC3147. LtnA and LtnB contain putative leader peptide sequences similar to the known `double glycine' type. The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the puri®ed a and b peptides in the residues serine, threonine, cysteine and alanine. Post-translational modi®cation, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference. Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters. In both cases, the gene clusters are ¯anked by IS946 elements, suggesting an en bloc transposition. The ®ndings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides. INTRODUCTION Lactic acid bacteria (LAB) produce several types of bacter- iocins. Comprehensive biochemical, structural and genetic characterization of LAB bacteriocins over the last few years has allowed the classi®cation of these substances (Klaenhammer 1993; Nes et al. 1996; Sahl and Bierbaum 1998). Major groups described differentiate between lanti- biotics (Class I) and small, heat-stable non-lantibiotics (Class II). Lantibiotics are post-translationally modi®ed peptides that exert a broad spectrum of inhibitory activity against other Gram-positive bacteria (Klaenhammer 1993). Nisin is the best characterized lantibiotic produced by LAB, and its use as a food preservative has been approved in several countries (Turtell and Delves-Broughton 1998). Most of the bacteriocins produced by LAB are included in class II, consisting of small, cationic and hydrophobic peptides. A peculiar group of these bacteriocins is charac- terized by the need for two peptides for full activity (Nissen-Meyer et al. 1992; Allison et al. 1994; Jime Ânez- Dõ Âaz et al. 1995; Marciset et al. 1997; Anderssen et al. 1998). A particular two-peptide bacteriocin system has been described in Enterococcus faecalis (cytolysins CylLL and CylLS) and Staphylococcus aureus C55 (staphylococcins C55a and C55b), where both peptides have been identi®ed as lantibiotics (Booth et al. 1996; Navaratna et al. 1998). The analysis of the complete sequence of the 60 kb conju- gative plasmid pMRC01 from L. lactis DPC3147 by Dougherty et al. (1998) revealed a genetic region of 20 kb, including an operon consisting of six genes responsible for the production of a two-peptide bacteriocin (lacticin 3147). Correspondence to: Dr T. Requena, Department of Dairy Science and Technology, Instituto del Frõ Âo (CSIC), Ciudad Universitaria, E-28040, Madrid, Spain (e-mail: trequena@if.csic.es). Journal of Applied Microbiology 2000, 89, 249260 = 2000 The Society for Applied Microbiology