Raffinocyclicin is a novel plasmid-encoded circular bacteriocin produced by Lactococcus raffinolactis with broad-spectrum activity against many gram-positive food pathogens

Abstract

This study describes the discovery and characterization of raffinocyclicin, a novel plasmid-encoded circular bacteriocin, produced by the raw milk isolate Lactococcus raffinolactis APC 3967. This bacteriocin has a molecular mass of 6,092 Da and contains 61 amino acids with a three-amino acid leader peptide. It shows the highest identity to the circular bacteriocins bacicyclicin XIN-1 (42.62%), aureocyclicin 4185 (42.62%), and garvicin ML (41.53%). A broad inhibitory spectrum includes strains from Staphylococcus, Enterococcus, Streptococcus, Micrococcus, Lactobacillus, Leuconostoc, and Listeria, in addition to a pronounced inhibitory effect against Lactococcus and Clostridium. It displays low sensitivity to trypsin, most likely as a result of its circular nature. The raffinocyclicin gene cluster is composed of 10 genes: 6 core genes, genes encoding an accessory three-component ABC transporter (rafCDE), and a putative transcriptional regulator related to the MutR family. A lack of inhibitory activity in the cell-free supernatant combined with the pronounced activity of cell extracts suggests that the majority of raffinocyclicin is associated with the cell rather than being released to the extracellular environment. This is the first report of a bacteriocin produced by the L. raffinolactis species.IMPORTANCEThe present study aimed to characterize raffinocyclicin, a novel circular bacteriocin produced by the lactic acid bacteria Lactococcus raffinolactis APC 3967. Bacteriocins are generally cationic and hydrophobic peptides with antimicrobial activity, which present diverse biotechnological properties of interest for the food industry. Raffinocyclicin inhibits a wide range of bacteria, including foodborne pathogens, and is stable against different treatments which suggest its potential as a natural biopreservative. Whole-genome sequencing and the genetic analysis of the raffinocyclicin gene cluster showed that it is encoded by plasmid that could be used in the future to transfer the ability to produce the bacteriocin to other lactic acid bacteria for industrial applications. These results together highlight the potential of this novel antimicrobial as a biopreservative to be used by the food industry.

Keywords: Lactococcus raffinolactis; antimicrobial activity; antimicrobial peptide; circular bacteriocin; raffinocyclicin.

Fig 1

Colony matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrum revealing the molecular mass of raffinocyclicin produced by L. raffinolactis APC 3967 (6,092 Da).

Fig 2

Genomic map of L. raffinolactis APC 3967. Chromosome (A); plasmid pRaff02 (B); plasmid pRaff03 (C); and plasmid pRaff01 (D). In green are all genes presented in the gene cluster of raffinocyclicin. The protein RepB is highlighted in red. The maps were created using Proksee (https://proksee.ca/).

Fig 3

Alignment of the precursor peptides of the putative bacteriocin and lactococcin 972, the leader sequences are underlined. Gap (−), identical amino acids (*), conservative substitutions (:) and weak conservative substitutions (.).

Fig 4

(A) Alignment of the precursor peptides of raffinocyclicin and garvicin ML, the leader sequences are underlined. Gap (−), identical amino acids (*), conservative substitutions (:), and weak conservative substitutions (.). The leader sequences are underlined. (B) The nucleotide sequence of the raffinocyclicin propeptide. The cleavage site is highlighted by the dashed line between residues 3 and 4. RBS, putative ribosome-binding site—7 bp upstream of the start codon. *, stop codon. (C) Predicted circular structure of raffinocyclicin. The yellow amino acids correspond to the leader sequence of the bacteriocin.

Fig 5

The proposed gene cluster of raffinocyclicin.

Fig 6

(A) Amino acid alignment of the mature circular bacteriocins (MAFFT v7.4.89). Gap (−), identical amino acids (*), and conservative substitutions (:). Dashed red lines are used to show the region of the peptide where most of the positively charged amino acids are found (H, K, and R). (B) Maximum likelihood phylogenetic tree of the mature circular bacteriocins using MAFFT for the alignment. Bootstrapped *1,000 replicates. The green boxes were used to highlight raffinocyclicin, garvicin ML, bacicyclicin XIN-1, and aureocyclicin 4185. *, net charge predicted for the bacteriocin linear form using GeneScript Peptide Property Tool.

Fig 7

(A) HPLC analysis of the cell extract of L. raffinolactis APC 3967 with fraction 74, highlighted by the red circle, and showing antimicrobial activity against L. lactis HP. (B) Detection of the theoretical mass of raffinocyclicin by MALDI-TOF mass spectrum from fraction 74. Expected mass: 6,092.17 Da (±1 Da).

Fig 8

(A) Approximate maximum likelihood phylogenetic tree of non-redundant genomes belonging to L. raffinolactis. The tree showing all 17 genomes (16 L. raffinolactis and 1 L. laudensis) was computed using a core gene alignment file generated using Roary, bootstrapped *1,000 replicates. The strain L. laudensis DSM 28961 was used as an outlier. (B) Raffinocyclicin gene clusters encoded by different L. raffinolactis strains.