Cloning and functional expression of a food-grade circular bacteriocin, plantacyclin B21AG, in probiotic Lactobacillus plantarum WCFS1

Abstract

There is an increasing consumer demand for minimally processed, preservative free and microbiologically safe food. These factors, combined with risks of antibiotic resistance, have led to interest in bacteriocins produced by lactic acid bacteria (LAB) as natural food preservatives and as potential protein therapeutics. We previously reported the discovery of plantacyclin B21AG, a circular bacteriocin produced by Lactobacillus plantarum B21. Here, we describe the cloning and functional expression of the bacteriocin gene cluster in the probiotic Lactobacillus plantarum WCFS1. Genome sequencing demonstrated that the bacteriocin is encoded on a 20 kb native plasmid, designated as pB21AG01. Seven open reading frames (ORFs) putatively involved in bacteriocin production, secretion and immunity were cloned into an E. coli/Lactobacillus shuttle vector, pTRKH2. The resulting plasmid, pCycB21, was transformed into L. plantarum WCFS1. The cell free supernatants (CFS) of both B21 and WCFS1 (pCycB21) showed an antimicrobial activity of 800 AU/mL when tested against WCFS1 (pTRKH2) as the indicator strain, showing that functional expression of plantacyclin B21AG had been achieved. Real-time PCR analysis revealed that the relative copy number of pB21AG01 was 7.60 ± 0.79 in L. plantarum B21 whilst pCycB21 and pTRKH2 was 0.51 ± 0.05 and 25.19 ± 2.68 copies respectively in WCFS1. This indicates that the bacteriocin gene cluster is located on a highly stable low copy number plasmid pB21AG01 in L. plantarum B21. Inclusion of the native promoter for the bacteriocin operon from pB21AG01 results in similar killing activity being observed in both the wild type and recombinant hosts despite the lower copy number of pCycB21.

Fig 1. Map of pTRKH2, pB21AG01 and pCycB21.

Top left: The plasmid pTRKH2 has an origin of replication from plasmid P15A and a replication gene (green arrows) for replication in both E. coli and Gram-positive bacteria; an erythromycin resistance gene (ErmR, yellow arrow) for selection in E. coli and LAB; and a multiple cloning site (MCS). Top right: Map of pB21AG01 containing 26 open reading frames, with seven ORFs corresponding to bacteriocin production, immunity and transportation (red arrows); a plasmid replication and relaxation gene (purple arrow); a transcription regulator (orange arrow), three conjugation transfer genes (blue arrows) and 14 other ORFs (grey arrows). Bottom: Map of pCycB21 harbouring seven bacteriocin-associated genes cloned into SacI and BamHI restriction sites.

Fig 2

Antimicrobial activity of a two-fold serial dilution of plantacyclin B21AG secreted by (a) Lactobacillus plantarum B21 and (b) WCFS1 harbouring pCycB21. L. plantarum WCFS1 (pTRKH2) was used as the indicator strain. Numbers above the wells correspond to the CFS dilution in each well. 1, Undiluted CFS; 2, 1:2 dilution of CFS; 3, 1:4 dilution of CFS; 4, 1:8 dilution of CFS; 5, 1:16 dilution of CFS; 6, 1:32 dilution of CFS; 7,1:64 dilution of CFS; 8, 1: 128 dilution of CFS. Well 9 (indicator strain) and well 10 (MRS broth) are negative controls.

Fig 3. MALDI-TOF-MS spectrum of plantacyclin B21AG.

A single peak was detected at molecular mass of 5664.69 for B21 and 5663.92 for WCFS1 (pCycB21). No major peaks were observed for WCFS1 (pTRKH2).