Purification and Characterization of a Novel Class IIa Bacteriocin, Piscicocin CS526, from Surimi-Associated Carnobacterium piscicola CS526

The bacteriocin piscicocin CS526 was inactivated by proteolyticenzymes, was stable at 100°C for 30 min, had a pH rangeof 2 to 8, and was active against Enterococcus, Listeria, Pediococcus,and Leuconostoc. The N-terminal sequence was YGNGL, not theYGNGV consensus motif common in class IIa bacteriocins (alternateresidues underlined). The molecular mass of piscicocin CS526,which had a bactericidal mode of action, was $~$ 4,430 Da.

INTRODUCTION

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Introduction

Bacteriocins are ribosomally synthesized proteins that inhibitbacteria closely related to the producer strain, food-bornepathogens, and spoilage bacteria (12, 18). Carnobacterium piscicolaCS526 inhibits Listeria monocytogenes growth in cold-smokedsalmon (21). Several bacteriocin-producing carnobacteria havebeen reported (1, 4, 10, 11, 13, 17). The objectives of thisstudy were to purify and characterize piscicocin CS526.

Preliminary characterization of the bacteriocin.

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Preliminary characterization of...

To determine the effects of enzymes on the bacteriocin, thecell supernatants (180 µl) of the culture neutralizedwith NaOH were incubated with 20 µl of enzyme solutions(10 mg/ml in 50 mM phosphate buffer [pH 7.0]) at 35°C for1 h. After boiling for 5 min, the residual activity was determinedby the agar well diffusion assay (21). The inhibitory activitywas completely destroyed by ${alpha}$ -chymotrypsin, papain, proteinaseK, and actinase and partly inhibited by trypsin (67% of residualactivity). Other enzymes, such as catalase, RNase, and lipase,did not affect the activity.

To determine the thermal stability, the cell supernatant ofthe neutralized culture was heated for 15 or 30 min at 70, 80,90, 100, and 121°C and then assayed for activity. The inhibitoryactivity was completely stable after 30 min at 100°C butwas partially inactivated at 121°C for 15 min (44% of residualactivity).

To determine the effect of pH on bacteriocin activity, 50 µlof the cell supernatant was added to 950 µl of trypticsoy broth with 0.6% yeast extract (TSBYE) adjusted with NaOHor HCl to different pH values. The samples were assayed foractivity after 1 h at 25°C. The activity was completelystable at pH 2 to 8 but reduced at pH values of 9 to 11 (67,44, and 30% of residual activity at pH 9, 10, and 11, respectively).

The antagonistic effect of cell supernatant of an overnightculture of CS526 in TSBYE against 27 strains of gram-positivebacteria and 6 strains of gram-negative bacteria was determinedby the agar well diffusion assay. Enterococcus faecalis JCM8726,Enterococcus faecium JCM8727, Enterococcus durans JCM8725, Enterococcushirae JCM8729, Pediococcus pentosaceus JCM5890, Tetragenococcushalophilus JCM5888, and Leuconostoc mesenteroides subsp. mesenteroidesJCM6124 were inhibited. L. monocytogenes IID578 and IID581,Listeria innocua FTHU8221, and Listeria grayi subsp. murrayiFTHU204 were also inhibited. Gram-positive spore-forming bacteria(Bacillus cereus JCM2152, Bacillus subtilis IFO13719 Bacillusmegaterium IAM13418 Bacillus licheniformis IFO12107 Bacillussphaericus IAM13420 and Paenibacillus polymyxa JCM2507) andgram-negative bacteria (Escherichia coli IFO15034 Salmonellaenterica serovar Enteritidis RIMD1933001, Pseudomonas fluorescensJCM5963m and Aeromonas hydrophila IFO3820) were not inhibited.These results demonstrated that the inhibitory activity of theC. piscicola CS526 was due to a proteinaceous molecule exhibitingexcellent heat stability. Therefore, the antilisterial substanceproduced by C. piscicola CS526 was confirmed as a bacteriocinand the bacteriocin has been named piscicocin CS526.

Mode of action.

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Mode of action.

The effect of the bacteriocin on nongrowing cells of L. monocytogenesIID581 and E. hirae JCM8729 was studied in TSBYE. Both strainswere grown at 30°C for 16 h in TSBYE, harvested by centrifugation,and resuspended in fresh TSBYE (pH 7.0) to $~$ 10⁶ cells/ml. Thefilter-sterilized cell supernatant of the C. piscicola CS526culture was immediately added to final concentrations of 600arbitrary units (AU)/ml, and the cells were incubated at 30°C.The viable cell counts of L. monocytogenes and E. hirae decreasedquickly from 8.0 x 10⁵ and 1.7 x 10⁶ CFU/ml to 1.5 x 10⁴ and1.4 x 10⁴ CFU/ml, respectively, after the treatment (30 min)with piscicocin CS526 (Fig. 1), indicating that piscicocin CS526is bactericidal rather than bacteriostatic.

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FIG. 1. Survivor curve of L. monocytogenes IID581 (circles) and E. hirae JCM8729 (triangles) in the presence of piscicocin CS526. The concentrations of the bacteriocin were 0 (open symbols) and 600 (closed symbols) AU/ml.

Bacteriocin purification and molecular properties.

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Bacteriocin purification and...

Bacteriocin was purified from the supernatant of a 2-liter C.piscicola CS526 culture propagated at 30°C for 12 h. Bacteriocinwas precipitated with ammonium sulfate (50%[wt/vol]), collectedby centrifugation (15,000 x g, 30 min, 4°C), and solubilizedin 20 mM sodium phosphate buffer (pH 6.0) (buffer A). The samplewas applied onto a 300-ml Sephadex G50 (Amersham Biosciences,Tokyo, Japan) gel-filtration gel equilibrated with buffer A,and the bacteriocin was eluted. The active fraction was appliedonto a 30-ml SP-Sepharose Fast Flow (Amersham Biosciences) cation-exchangecolumn equilibrated with 25 mM sodium N-morpholinoethanesulfonicacid (MES) containing 3.0 M urea, pH 6.0. (buffer B). Afterthe column was washed with 120 ml of buffer B, the bacteriocinwas eluted with linear NaCl gradient (0 to 0.5 M) at pH 6.0,and loaded on a Sep-Pack C₁₈ cartridge (Waters, Milford, Mass.).The cartridge was washed with 20% 2-propanol in 0.1% trifluoroaceticacid (TFA), and the bacteriocin was eluted with 80% 2-propanolin 0.1% TFA. After drying, the bacteriocin was dissolved in0.05% TFA and applied to C₁₈ reverse-phase column (ODS-80Ts;4.6 by 150 mm; Tosho, Tokyo, Japan) connected to a high-performanceliquid chromatograph. Elution was performed by using a lineargradient from 100% 0.05 TFA to 100% 2-propanol in 60 min ata flow rate of 0.5 ml/min.

The purification was approximately a 1.6 x 10⁴-fold overall,with a 7% yield. The inhibitory fraction after reverse-phasehigh-performance liquid chromatography (RP-HPLC) was analyzedfurther by Tricine sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) methods (5). SDS-PAGE revealed asingle band of protein (Fig. 2D, lane 2) that inhibited L. monocytogenesIID581 (Fig. 2D, lane 3). It was estimated to be a 4.0- to 4.5-kDapeptide.

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FIG. 2. Purification of piscicocin CS526 by gel filtration (A), cation-exchange (B) and RP-HPLC (C), and antibacterial detection SDS-PAGE of piscicocin CS526 (D). Purified piscicocin CS526 was analyzed by SDS-PAGE. The gel was removed and cut into two parts. The first half, containing molecular weight markers (lane 1) and the purified piscicocin CS526 (lane 2), was stained with silver. The other half, containing the purified bacteriocin (lane 3), was overlaid with L. monocytogenes IID581 and incubated at 30°C for 24 h. The arrows indicate the bacteriocin activities.

The purified piscicocin CS526 was subjected to mass spectralmeasurement by electrospray ionization spectrometry (model JM-700TZmass spectrometer); JEOL, Peabody, Mass. Two unequivocal pseudomolecularions [M + nH]ⁿ⁺ were observed at m/z corresponding to n = 2and 3 protonated species, whose averaged molecular mass was4,429.6 Da (Fig. 3).

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FIG. 3. Electrospray ionization mass spectrometry analysis of purified piscicocin CS526.

The partial NH₂-terminal amino acid sequence, comprising 43amino acid residues, was determined by Edman degradation withProcise 492-HT Protein Sequencer (Applied Biosystems, FosterCity, Calif.) (Table 1). Piscicocin CS526 shared significanthomology with munditicin and piscicolin 126, produced, respectively,by Enterococcus mundtii (3) and C. piscicola JG126 (11), belongingto class IIa bacteriocins. However, there were no identicalsequences in the database. Class IIa bacteriocins, accordingto the system proposed by Klaenhammer (12), have antilisterialactivity and possess the highly conserved N-terminal motif,YGNGV (15). Bhugaloo-Vial et al. (4) also indicated that itwas possible to increase the consensus sequence as YGNGVXCX(K/N)XXCXV(N/D)(W/K/R)X(G/A/S)(A/N).Piscicocin CS526 exhibited strong antilisterial activity butpossessed a novel N-terminal sequence that contained a changeof Val to Leu at position 7. Only three other bacteriocins,bacteriocin 31 (19), sakacin 5X (20), and plantaricin C19 (2),possess this altered N-terminal sequence. The YGNGV consensusmotif has been involved in a recognition step of the mechanismof action of class IIa bacteriocins (7, 8), but the YGNGV consensusmotif did not appear to be involved in the initial binding stepfor pediocin PA-1 (6) Carnobacteriocin B2 activity was reducedby a substitution within the Y³GNGV motif (Tyr³ to Phe) (16).Pediocin AcH activity was dramatically reduced by a mutationwithin the YGN⁵GV motif (Asn⁵ to Lys) (14). In this study, wefound that piscicocin CS526, which possessed YGNGL sequence,had anti-Listeria activity typical of class IIa bacteriocins.Accordingly, the presence of Val⁷ within the YGNGV⁷ motif couldnot be a prerequisite for the antimicrobial activity of classIIa bacteriocins.

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TABLE 1. Sequence alignment of piscicocin CS526 produced by C. piscicola CS526 and other class IIa bacteriocins

FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Marine Food Science, Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate 041-8611, Japan. Phone: 81-138-40-5574. Fax: 81-138-40-5573. E-mail: yamasaki{at}fish.hokudai.ac.jp.

REFERENCES

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