Discovery and characterisation of novel circular bacteriocin plantacyclin B21AG fromLactobacillus plantarumB21

Abstract

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.

Cloning and functional expression of a food-grade circular bacteriocin, plantacyclin B21AG, in probioticLactobacillus plantarumWCFS1

“…Seven ORFs were predicted to encode genes putatively responsible for the production, immunity and transport of plantacyclin B21AG ( orf19 – orf25 ) [30]. Orf19 showed 88 % identity to Plantaricyclin A precursor (PlcA), a circular bacteriocin produced by L. plantarum (PCL98053.1), presumably the structural gene responsible for the production of plantacyclin B21AG.…”

“…Several strategies for heterologous expression of bacteriocins have been investigated either for overproduction of the bacteriocin or structure-function studies [7,27–29]. We previously reported the discovery of plantacyclin B21AG, a food-grade circular bacteriocin produced by Lactobacillus plantarum B21 [30,31]. It is shown to be active against food-borne pathogens including Clostridium perfringens and Listeria monocytogenes ; food spoilage bacteria such as L. arabinosus ; as well as other LAB including L. plantarum , L. brevis and Lactococcus lactis [30–32].…”

“…We previously reported the discovery of plantacyclin B21AG, a food-grade circular bacteriocin produced by Lactobacillus plantarum B21 [30,31]. It is shown to be active against food-borne pathogens including Clostridium perfringens and Listeria monocytogenes ; food spoilage bacteria such as L. arabinosus ; as well as other LAB including L. plantarum , L. brevis and Lactococcus lactis [30–32]. This study aimed to transfer the production of the broad antimicrobial spectrum of plantacyclin B21AG to a probiotic strain, L. plantarum WCFS1 [33].…”

Crystal structure and site-directed mutagenesis of circular bacteriocin plantacyclin B21AG reveals cationic and aromatic residues important for antimicrobial activity

“…These bacteriocins are found across a wide range of Grampositive genera. Family II are generally found in Lactobacillus, Staphylococcus and Streptococcus species, and includes gassericin A/reutericin 6 26 , butyrivibriocin AR10 27 , acidocin B 28 , paracyclicin 29 , plantaricyclin A 30 and plantacyclin B21AG [31][32][33] .…”

“…We previously identified a circular bacteriocin plantacyclin B21AG from Lactobacillus plantarum B21. This family II circular bacteriocin has antimicrobial activity against foodborne pathogens including Listeria monocytogenes and Clostridium perfringens and other closely related Lactobacillus species 31 . We have also cloned the plantacyclin B21AG gene cluster into a plantacyclin B21AG-negative strain and used this system for its expression 42 .…”