Production of pediocin PA-1 by Lactococcus lactis using the lactococcin A secretory apparatus

Abstract

The class II bacteriocins pediocin PA-1, from Pediococcus acidilactici, and lactococcin A, from Lactococcus lactis subsp. lactis bv. diacetylactis WM4 have a number of features in common. They are produced as precursor peptides containing similar amino-terminal leader sequences with a conserved processing site (Gly-Gly at positions -1 and -2). Translocation of both bacteriocins occurs via a dedicated secretory system. Because of the strong antilisterial activity of pediocin PA-1, its production by lactic acid bacteria strains adapted to dairy environments would considerably extend its application in the dairy industry. In this study, the lactococcin A secretory system was adapted for the expression and secretion of pediocin PA-1. A vector containing an inframe fusion of sequences encoding the lcnA promoter, the lactococcin A leader, and the mature pediocin PA-1, was introduced into L. lactis IL1403. This strain is resistant to pediocin PA-1 and encodes a lactococcin translocation apparatus. The resulting L. lactis strains secreted a bacteriocin with an antimicrobial activity of approximately 25% of that displayed by the parental pediocin-producing P. acidilactici 347. A noncompetitive indirect enzyme-linked immunosorbent assay with pediocin PA-1-specific antibodies and amino-terminal amino acid sequencing confirmed that pediocin PA-1 was being produced by the heterologous host.

FIG. 1

PCR strategy used for splicing the lacA and pedA genes. (a) Region of the lactococcin A operon, containing lcnA and lciA genes, cloned into pFI2058. (b) Map of the pedA gene. The thick arrows show coding regions with the amino-terminal leaders indicated in black. The small arrows above and below the maps indicate the position and direction of primers used for PCR. Nonhomologous tails on primers plcnF and ppepE are represented by dashed lines. (c) Fragments generated by the first PCR step. (d) Homologous regions in fragments 1 and 2 annealed to provide a template for the second PCR step, involving primers plcn1 and ppepD. (e) Product of the second PCR step, cloned into pFI2126. An in-frame fusion resulted in the creation of a consensus amino-terminal cleavage site (vertical arrow) between two parts of the lcnA/pedA hybrid gene.

FIG. 2

Agar diffusion bioassay for detection of bacteriocin activity against E. faecium P21. 1 and 5, L. lactis FI9043 (MRS culture); 2 and 6, L. lactis FI9043 (GM17 culture); 3, P. acidilactici 347; 4, L. lactis IL1403.

FIG. 3

Reverse-phase chromatography of pediocin PA-1. The amount applied to the column was obtained from a 1-liter culture of L. lactis FI9043. BU, bacteriocin units.

FIG. 4

Standard curves for pediocin PA-1 purified from P. acidilactici 347 (▪) and L. lactis FI9043 (○), OA-PH2 (□), nisin (•), and the protein fraction obtained from culture supernatants of P. acidilactici 347-8, a P. acidilactici 347-cured derivative that does not produce pediocin PA-1 (⧫), as determined by NCI-ELISA with anti-pediocin PA-1 rabbit antibodies. Each datum point represents the average value of triplicate determinations in a single microtiter plate.