Plantaricyclin A, a Novel Circular Bacteriocin Produced by Lactobacillus plantarum NI326: Purification, Characterization, and Heterologous Production

Abstract

Bacteriocins from lactic acid bacteria (LAB) are of increasing interest in recent years due to their potential as natural preservatives against food and beverage spoilage microorganisms. In a screening study for LAB, we isolated from olives a strain, Lactobacillus plantarum NI326, with activity against the beverage-spoilage bacterium Alicyclobacillus acidoterrestris Genome sequencing of NI326 enabled the identification of a gene cluster (designated plc) encoding a putative circular bacteriocin and proteins involved in its modification, transport, and immunity. This novel bacteriocin, named plantaricyclin A (PlcA), was grouped into the circular bacteriocin subgroup II due to its high degree of similarity with other gassericin A-like bacteriocins. Purification of PlcA from the supernatant of Lb. plantarum NI326 resulted in an active peptide with a molecular mass of 5,570 Da, corresponding to that predicted from the (processed) PlcA amino acid sequence. The plc gene cluster was cloned and expressed in Lactococcus lactis NZ9000, resulting in the production of an active 5,570-Da bacteriocin in the supernatant. PlcA is believed to be produced as a 91-amino-acid precursor with a 33-amino-acid leader peptide, which is predicted to be removed, followed by joining of the N and C termini via a covalent linkage to form the mature 58-amino-acid circular bacteriocin PlcA. We report the characterization of a circular bacteriocin produced by Lb. plantarum The inhibition displayed against A. acidoterrestris highlights its potential use as a preservative in food and beverages.IMPORTANCE In this work, we describe the purification and characterization of an antimicrobial peptide, termed plantaricyclin A (PlcA), produced by a Lactobacillus plantarum strain isolated from olives. This peptide has a circular structure, and all genes involved in its production, circularization, and secretion were identified. PlcA shows antimicrobial activity against different strains, including Alicyclobacillus acidoterrestris, a common spoilage bacterium, which causes substantial economic losses in the beverage industry every year. In this study, we describe a circular antimicrobial peptide, PlcA, for a Lactobacillus plantarum strain.

Keywords: Alicyclobacillus acidoterrestris; Lactobacillus plantarum; circular bacteriocin; immunity.

FIG 1

(A) Sequence alignment of all the members of subgroup II circular bacteriocins with plantaricyclin A, using MUSCLE (41). Conserved, conservative, and semiconservative substitutions are indicated by asterisks, colons, and semicolons, respectively. Bold letters depict the leader sequence. (B) Schematic plantaricyclin A mature peptide.

FIG 2

Schematic representation of the gene clusters involved in the production of the circular bacteriocins gassericin A (24), acidocin B (18) and plantaricyclin A. The known or putative biochemical function or properties are denoted by color, as indicated in the key.

FIG 3

(A) Antimicrobial activity of the CFS of L. plantarum NI326 and nisin A-induced L. lactis NZ9000(pNZPlc) against A. acidoterrestris sp1. (B) Antimicrobial activity of the CFS of Lb. plantarum NI326 against cultures of L. lactis NZ9000(pNZ8048), L. lactis NZ9000(pNZPlcD), L. lactis NZ9000(pNZPlcI), and L. lactis NZ9000(pNZPlcDI) uninduced (−) or induced (+) with nisin A.

FIG 4

MALDI-TOF mass spectrometry analysis of the purified plantaricyclin A produced by L. lactis pNZPlca (A) and L. plantarum NI326 (B). %Int., percentage of intensity.