Toyoncin, a Novel Leaderless Bacteriocin That Is Produced by Bacillus toyonensis XIN-YC13 and Specifically Targets B. cereus and Listeria monocytogenes

Abstract

Bacteriocins have attracted increasing interest because of their potential as natural preservatives. Recent studies showed that the Bacillus cereus group is a prominent producer of bacteriocins. Using a laboratory-based screening strategy, we identified a strain in the B. cereus group, Bacillus toyonensis XIN-YC13, with antimicrobial activity against B. cereus. A novel, 70-amino-acid-long leaderless bacteriocin, toyoncin, was purified from the culture supernatant of strain XIN-YC13, and its molecular mass was found to be 7,817.1012 Da. Toyoncin shares no similarity with any other known bacteriocins, and its N-terminal amino acid is formylmethionine rather than methionine. Toyoncin shows good pH and heat stability and exhibits specific antimicrobial activity against two important foodborne pathogens, B. cereus and Listeria monocytogenes. Additionally, toyoncin exerts bactericidal activity and induces cell membrane damage. Toyoncin can also inhibit the outgrowth of B. cereus spores. Preservation assays showed that toyoncin effectively suppressed or eradicated B. cereus and L. monocytogenes in pasteurized skim milk. These results suggest that toyoncin can be used as a new biopreservative against B. cereus and L. monocytogenes in the food industry. IMPORTANCE We identified a novel leaderless bacteriocin, toyoncin, produced by B. toyonensis XIN-YC13. Toyoncin shows good pH and heat stability, and it has specific antimicrobial activity against B. cereus and L. monocytogenes (two important foodborne pathogens), likely by destroying their cell membrane integrity. Toyoncin inhibited the outgrowth of B. cereus spores and effectively inhibited or eliminated B. cereus and L. monocytogenes in a milk model system. These results indicate the potential of toyoncin as a food preservative.

Keywords: Bacillus toyonensis; bacteriocin; food preservatives; toyoncin.

FIG 1

Screening the bacteriocin-producing strain Bacillus toyonensis XIN-YC13. (A) Colony morphology of B. toyonensis XIN-YC13. (B) Growth kinetics of B. toyonensis XIN-YC13 in Luria-Bertani (LB) medium. Experiments were performed in triplicate, and data are shown as mean values ± standard deviations (SD). (C) Antimicrobial activity of the supernatant of B. toyonensis XIN-YC13 against B. cereus ATCC 14579. The untreated supernatant was added to the outer well, and the corresponding supernatant treated with mixed enzymes was added to the inner well.

FIG 2

Purification and identification of toyoncin. (A) HPLC analysis of crude extracts of B. toyonensis XIN-YC13. The effluent was monitored at 220 nm and manually collected every minute. (B) Q-TOF mass spectrometry of the HPLC-purified toyoncin. M.W., molecular weight.

FIG 3

Amino acid sequences and putative biosynthetic gene clusters of toyoncin and its analogues. (A) Sequence alignment of the precursor peptide of toyoncin (ToyA) with the precursor peptides of its analogues. The underlined characters represent the 39 N-terminal amino acids of deformylated toyoncin sequenced by Edman degradation. The N-terminal amino acid of ToyA, methionine, is formylated (marked with an asterisk) to form the mature bacteriocin product toyoncin. (B) Gene clusters of toyoncin and its analogues were distributed in four Bacillus toyonensis strains. Open reading frames (ORFs) are colored according to their predicted functions. *, analyses were performed using the BLASTP program, and the proteins listed were the closest homologue to each ORF in the putative biosynthetic gene cluster of toyoncin in the GenBank database.

FIG 4

Effects of pH, temperature, and proteases on antimicrobial activity of toyoncin. (A) Stability of toyoncin at 37°C or 65°C under different pH conditions. All tests were performed in triplicate, and data are shown as mean values ± SD. (B) Effects of various proteases on toyoncin.

FIG 5

Toyoncin exerts bactericidal activity and causes membrane damage. (A) Effects of toyoncin on the number of viable cells and optical density of B. cereus ATCC 14579 culture. The data are shown as the mean values and SD. (B) SEM analysis of toyoncin-treated B. cereus cells. (C) Flow cytometry analysis of cell membrane damage by PI uptake staining. B. cereus cells were exposed to 0-, 2-, and 4-fold MIC of toyoncin for 2 h. The percentage of PI uptake by cells was analyzed with a flow cytometer. The data are shown as the mean values and SD. (D) Effect of toyoncin on amount of potassium released from B. cereus cells. The data are shown as the mean values and SD.

FIG 6

Toyoncin inhibits the outgrowth of Bacillus cereus spores. Black arrows indicate spores of B. cereus ATCC 14579. Red arrows indicate vegetative cells of B. cereus ATCC 14579. Scale bars represent 5.0 μm.

FIG 7

Preservative effect of toyoncin against B. cereus (A) and L. monocytogenes (B) in pasteurized skim milk. Pasteurized milk samples with B. cereus and L. monocytogenes were stored at 4°C for 14 days. Commercial nisin A (1,000 U) was used as a positive control, and ddH₂O was used as a negative control. All tests were performed in triplicate, and data are presented as mean values ± SD.