Lactobacillus plantarum B21 isolated from Vietnamese sausage (nem chua) has previously displayed broad antimicrobial activity against gram positive bacteria including foodborne pathogens Listeria monocytogenes and Clostridium perfringens. This study successfully identified the antimicrobial agent as plantacyclin B21AG, a 5668 Da circular bacteriocin demonstrating high thermostability, resistance to a wide range of pH, proteolytic resistance and temporal stability. We report a reverse genetics approach used to identify and characterise plantacyclin B21AG. The bacteriocin was purified from culture supernatant by a short process consisting of concentration, n-butanol extraction and cation exchange chromatography. A de novo peptide sequencing using LC-MS/MS techniques identified two putative peptide fragments which were mapped to the genome of Lactobacillus plantarum B21. This revealed an ORF corresponding to a putative circular bacteriocin with a 33-amino acid leader peptide and 58-amino acid mature peptide found on native plasmid pB21AG01. The corresponding gene cluster, consisted of seven genes associated with post-translational circularisation, immunity and secretion. The robust nature of plantacyclin B21AG, its antimicrobial activity and associated machinery for cyclisation make it an interesting biotechnological target for further development, and application as a food-safe antimicrobial.
Lactiplantibacillus plantarum B21 isolated from Vietnamese sausage ( nem chua ) has previously displayed broad antimicrobial activity against Gram-positive bacteria including foodborne pathogens Listeria monocytogenes and Clostridium perfringens . This study successfully identified the antimicrobial agent as plantacyclin B21AG, a 5668 Da circular bacteriocin demonstrating high thermostability, resistance to a wide range of pH, proteolytic resistance and temporal stability. We report a reverse genetics approach to identify and characterise plantacyclin B21AG from first principles. The bacteriocin was purified from culture supernatant by a three-step process consisting of concentration, n-butanol extraction and cation exchange chromatography. A de novo peptide sequencing using LC-MS/MS techniques identified two putative peptide fragments which were mapped to the genome sequence of L. plantarum B21. This revealed an ORF corresponding to a putative circular bacteriocin with a 33-amino acid leader peptide and a 58-amino acid mature peptide encoded on a native plasmid pB21AG01. The bacteriocin is shown to be a small cationic predominantly α-helical protein (69%). The corresponding gene cluster, consisted of seven genes associated with post-translational circularisation, immunity and secretion. Whilst plantacyclin B21AG is 86% identical to the newly published plantaricyclin A it is more highly cationic having a net charge of +3 due to an additional basic residue in the putative membrane interaction region. This and other substitutions may well go some way to explaining functional differences. The robust nature of plantacyclin B21AG, its antimicrobial activity and associated machinery for cyclisation make it an interesting biotechnological target for development, both as a food-safe antimicrobial or potentially a platform technology for recombinant protein circularisation.
Lactobacillus plantarum strain B21 was isolated from Vietnamese sausage (nem chua) and demonstrated broad antimicrobial activity due to the production of bacteriocins. Here, we report the complete genome sequence of this strain (3,284,260 bp).
25There is an increasing consumer demand for minimally processed, preservative free and 26 microbiologically safe food. These factors, combined with risks of antibiotic resistance, have 27 led to interest in bacteriocins produced by lactic acid bacteria (LAB) as natural food 28 preservatives and as protein therapeutics. We previously reported the discovery of 29 plantacyclin B21AG, a novel circular bacteriocin produced by Lactobacillus plantarum B21. 30 Here, we describe the cloning and functional expression of the bacteriocin gene cluster in the 31 probiotic Lactobacillus plantarum WCFS1. Genome sequencing demonstrated that the 32 bacteriocin is encoded on a 20 kb native plasmid, designated as pB21AG01. Seven open 33 reading frames (ORFs) putatively involved in bacteriocin production, secretion and immunity 34 were cloned into an E. coli/Lactobacillus shuttle vector, pTRKH2. The resulting plasmid, 35 pCycB21, was transformed into L. plantarum WCFS1. The cell free supernatants (CFS) of 36 both B21 and WCFS1 (pCycB21) showed an antimicrobial activity of 800 AU/mL when 37 tested against the WCFS1 (pTRKH2) indicator strain, indicating functional expression of 38 plantacyclin B21AG. Real-time PCR analysis revealed that the relative copy number of 39 pB21AG01 was 7.60 + 0.79 in L. plantarum B21 whilst pCycB21 and pTRKH2 was 0.51 + 40 0.05 and 25.19 + 2.68 copies, respectively in WCFS1. This indicates that the bacteriocin gene 41 cluster is located on a highly stable, low copy number plasmid pB21AG01 in L. plantarum 42 B21. Inclusion of the native promoter for the bacteriocin operon from pB21AG01 may result 43 in similar inhibitory zones observed in both wild type and recombinant hosts despite the low 44 copy number of pCycB21.45 46 Bacteriocins are ribosomally synthesised, extracellularly released peptides or peptide 48 complexes that possess antibacterial activity against species usually closely related to the 49 producer strains or a wider range of microorganisms [1,2]. Interestingly, the bacteriocins 50 produced by gram-positive bacteria seem to exhibit broader spectrum activity compared to 51 the gram-negative bacteria [3]. Among the gram-positive bacteria, bacteriocins produced by 52 the food-grade lactic acid bacteria (LAB) have attracted considerable interest because they 53 are generally regarded as safe (GRAS). Being proteins they can be easily degraded by 54 proteases in the mammalian gastrointestinal tract, making them safe for human consumption 55 and minimizing the risk of developing resistant bacteria [4,5]. They have been widely used as 56 natural food preservatives for controlling food-borne and food-spoilage bacteria without 57 affecting sensory qualities. They also have huge potential in veterinary applications and as 58 next-generation antibiotics against multi-drug resistant (MDR) pathogens [5-7]. One of the 59 advantages of bacteriocins over conventional antibiotics is that they are directly gene 60 encoded, making bioengineering feasible to enhance their productivity or specificity towards 61 ...
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