A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial ␣-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1.As a consequence of recurring and serious listeriosis outbreaks (5, 11), Listeria monocytogenes has become a focus of bacteriocin researchers. The search for bacteriocin-producing lactic acid bacteria has been directed towards substances whose targets are Listeria spp., and consequently, a large number of antilisterial bacteriocins have been revealed (2,7,12). Particularly, class IIa bacteriocins (pediocin-like bacteriocins) are highly active against Listeria spp. and are the most promising candidates for food biopreservatives (4). Pediocin PA-1, produced by Pediococcus spp., is a representative class IIa bacteriocin and has been heterologously produced by other genera to inhibit food-borne pathogens or food spoilage bacteria (6,14,17). However, to the best of our knowledge, study of the heterologous production of pediocin PA-1 in bifidobacteria has been absent. Bifidobacteria, the main microflora of the human intestinal tract (9), have been used as representative probiotics. Because bifidobacteria are generally recognized as safe and colonize the large intestine, they could be promising candidates as hosts for expression of beneficial foreign proteins/peptides for human use (8,16). In this study, pediocin PA-1 was heterologously expressed in Bifidobacterium longum MG1 and secreted from the strain by using the signal sequence for bifidobacterial ␣-amylase. The recombinant B. longum MG1 efficiently killed L. monocytogenes in a coculture.Escherichia coli JM109 was grown in LB medium at 37°C. B. longum MG1 was grown in MRS medium (Merck, Darmstadt, Germany) supplemented with 0.05% (wt/vol) L-cystein HCl (Sigma Co.) at 37°C without agitation. Lactobacillus plantarum NCDO 955 was grown in MRS medium at 37°C without agitation. L. monocytogenes KFRI 799 was grown in BHI medium at 35°C and on Oxford Listeria selective agar for the selective enumeration. Agar plates were made by adding 1.5% agar to broth media. Antibiotics (Sigma Co.) were added as selective agents with appropriate concentrations (chloramphenicol, 2 /ml for B. longum and 20 /ml for E. coli; ampicillin, 100 /ml). MRS agar plates spread with B. longum were anaerobically incubated in an atmosphere generation system (GasPak system, Oxoid, Basingstoke, Hampshire, England).Plasmids and PCR product used in this study are summarized in Table 1. Primers PSamyF (5Ј-GCTCTAGAGCGGG CATCGCCGAATATACTCCC-3Ј) and PSamyR (5Ј-GGCCT GTGCTGCGGTGCTGGC-3Ј) were used to amplify a 400-bp fragment containing a promoter and deduced signal sequence of the bifidobacterial ␣-amylase gene. A recombinant DNA, pBESAF2, was used as the template. Primers pedSF (5Ј-AAA TACTACGGTAATGGGGTTAC-3Ј) and pedABR (5Ј-CGG GATCCCGAAAAAGCCGCAAG...