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.
Highlights
Bacteriocins are ribosomally synthesised, extracellularly released peptides or peptide complexes that possess antibacterial activity against species usually closely related to the producer strains or a wider range of microorganisms [1, 2]
We previously reported the discovery of plantacyclin B21AG, a food-grade circular bacteriocin produced by Lactobacillus plantarum B21 [30, 31]
For the purpose of this study, we focused our analysis on pB21AG01 as it was found to encode the genes responsible for the production of a circular bacteriocin
Summary
Bacteriocins are ribosomally synthesised, extracellularly released peptides or peptide complexes that possess antibacterial activity against species usually closely related to the producer strains or a wider range of microorganisms [1, 2]. Amongst the Gram-positive bacteria, bacteriocins produced by the foodgrade lactic acid bacteria (LAB) have attracted considerable interest because they are generally regarded as safe (GRAS). Being proteins, they can be degraded by proteases in the mammalian gastrointestinal tract, making them safe for human consumption and minimizing the risk of developing resistant bacteria [4, 5]. They can be degraded by proteases in the mammalian gastrointestinal tract, making them safe for human consumption and minimizing the risk of developing resistant bacteria [4, 5] They have been widely used as natural food preservatives for controlling food-borne and food-spoilage bacteria without affecting food sensory qualities. One of the advantages of bacteriocins over conventional antibiotics is that they are directly gene encoded, making bioengineering and evolutionary approaches to tailor and enhance their action against specific pathogens entirely feasible [5, 8]
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