Leuconostoc lactis strain APC 3969 produces a new variant of cyclic bacteriocin leucocyclicin Q and displays potent anti-Clostridium perfringens activity

Abstract

Clostridium perfringens is an important foodborne pathogen that produces diverse toxins and is often associated with foodborne gastroenteritis. In this sense, novel biopreservatives with anti-C. perfringens activity are of interest. Among them, bacteriocins produced by lactic acid bacteria stand out as potential candidates. This study describes leucocyclicin C, a novel variant of the bacteriocin leucocyclicin Q, capable of inhibiting C. perfringens. The bacteriocin comprises 61 amino acids, has a molecular mass of 6,081.44 Da, and is produced by the strain Leuconostoc lactis APC 3969. Like many circular bacteriocins, leucocyclicin C has a broad spectrum of activity, is protease resistant, and has high stability against thermic and pH stresses. The leucocyclicin C genetic cluster comprises ten genes instead of the five genes previously described for leucocyclicin Q. Also, this genetic cluster seems to be part of a putative composite transposon. Leucocyclicin C has a minimum inhibitory concentration (MIC) of 3.288 µM against C. perfringens, comparable with other antimicrobial peptides. These results suggest that leucocyclicin C has the potential as a biopreservative for controlling C. perfringens in food.

Keywords: Clostridium perfringens; Leuconostoc lactis; Circular bacteriocin; Leucocyclicin C; Variant.

Fig. 1

Colony MALDI-TOF mass spectrum of the bacteriocin producing strain L. lactis APC 3969. The highest intensity peak (6,081.45 Da) is highlighted by the red circle.

Fig. 2

Alignment of the precursor peptides of the putative bacteriocins with lactococcin 972 (A), enterocin Xα (B), enterocin Xβ (C). The leader sequences are highlighted in yellow and the recognition sites for cleavage are highlighted in bold.

Fig. 3

(A) Alignment of the precursor peptides of leucocyclicin Q and leucocyclicin C showing the leader sequences are highlighted in yellow and a single threonine to alanine substitution in the structural peptide highlighted in blue. Identical amino acids (*) and conservative substitutions (:). (B) The nucleotide sequence of the leucocyclicin C propeptide. The cleavage site is highlighted by the dashed line between residues 2 and 3. RBS, putative ribosome binding site – 7 bp upstream of the start codon. *—stop codon.

Fig. 4

Gene clusters of leucocyclicin C and leucocyclicin Q. Data obtained from GenBank (AB795997.1) for leucocyclicin Q.

Fig. 5

Putative composite transposon that encodes the leucocyclicin C genetic cluster. The leucocyclicin C gene cluster is highlighted by a dashed green box. The inverted repeats are highlighted by a dashed blue box.

Fig. 6

(A) HPLC analysis of the cell extract of Leuconostoc lactis APC 3969 with fraction 51, highlighted by the red circle. (B) MALDI-TOS MS of fraction 51 showing a mass of 6081.06 Da which correlates with the theoretical mass of leucocyclocin C (6081.44 Da). Note that the 3043 Da mass is doubly charged leucocyclin C.

Fig. 7

Effect of different concentrations of leucocyclicin C on the growth of Clostridium perfringens EM124. The MIC value for the strain is highlighted by the blue circle.