Characterization and identification of weissellicin Y and weissellicin M, novel bacteriocins produced by Weissella hellenica QU 13

Authors

  • Y. Masuda,

    1.  Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Higashi-ku, Fukuoka, Japan
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  • T. Zendo,

    1.  Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Higashi-ku, Fukuoka, Japan
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  • N. Sawa,

    1.  Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Higashi-ku, Fukuoka, Japan
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  • R.H. Perez,

    1.  Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Higashi-ku, Fukuoka, Japan
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  • J. Nakayama,

    1.  Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Higashi-ku, Fukuoka, Japan
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  • K. Sonomoto

    1.  Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Higashi-ku, Fukuoka, Japan
    2.  Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, Higashi-ku, Fukuoka, Japan
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Kenji Sonomoto, Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
E-mail: sonomoto@agr.kyushu-u.ac.jp

Abstract

Aims:  To identify and characterize novel bacteriocins from Weissella hellenica QU 13.

Methods and Results: Weissella hellenica QU 13, isolated from a barrel used to make Japanese pickles, produced two novel bacteriocins termed weissellicin Y and weissellicin M. The primary structures of weissellicins Y and M were determined, and their molecular masses were determined to be 4925·12 and 4968·40 Da, respectively. Analysis of the DNA sequence encoding the bacteriocins revealed that they were synthesized and secreted without N-terminal extensions such as leader sequences or sec signal peptides. Weissellicin M showed significantly high and characteristic homology with enterocins L50A and L50B, produced by Enterococcus faecium L50, while weissellicin Y showed no homology with any other known bacteriocins. Both bacteriocins showed broad antimicrobial spectra, with especially high antimicrobial activity against species, which contaminate pickles, such as Bacillus coagulans, and weissellicin M showed relatively higher activity than weissellicin Y. Furthermore, the stability of weissellicin M against pH and heat was distinctively higher than that of weissellicin Y.

Conclusions: Weissella hellenica QU 13 produced two novel leaderless bacteriocins, weissellicin Y and weissellicin M, and weissellicin M exhibited remarkable potency that could be employed by pickle-producing industry.

Significance and Impact of the Study:  This study is the first report, which represents a complete identification and characterization of novel leaderless bacteriocins from Weissella genus.

Introduction

Biopreservation is the use of natural or controlled micro-organisms to preserve foods or extending their shelf life, and fermented foods can be regarded as typical model of biopreservation. Many micro-organisms grow in these foods and contribute to taste, flavour and preservation. Lactic acid bacteria (LAB) play an important role in producing various antimicrobial substances that eliminate other micro-organisms competing in the fermentation process (Caplice and Fitzgerald 1999). Some LAB are known to produce various types of bacteriocins, which are antimicrobial peptides that exhibit bactericidal effects against Gram-positive bacteria including foodborne pathogens but not against eukaryotic cells, and have a high stability against high temperature and low pH (Cotter et al. 2005; De Vuyst and Leroy 2007). These beneficial characteristics have led us to utilize bacteriocins as biopreservatives and bacteriocin-producing LAB as starter cultures for the fermentation of foods (Cleveland et al. 2001; Galvez et al. 2007).

LAB bacteriocins are mainly classified into two classes (Klaenhammer 1993; Nes and Holo 2000; Cotter et al. 2005). Class I bacteriocins, so-called lantibiotics, are heat-stable post-translationally modified peptides containing multiple rings bridged by lanthionine or 3-methyllanthionine residues (McAuliffe et al. 2001). Class II bacteriocins are small, heat-stable nonlantibiotic peptides and are further divided into four subgroups (Nes et al. 1996; Nes and Holo 2000). Class IIa bacteriocins are Listeria-active peptides with a consensus YGNGVXC sequence at the N-terminal (Ennahar et al. 2000). Class IIb bacteriocins comprise two peptides, both of which are required for complete antimicrobial activity. Class IIc bacteriocins are circular bacteriocins such as lactocyclicin Q (Sawa et al. 2009). Class IId bacteriocins are the other class II bacteriocins, including enterocin P, which is processed and secreted by the sec pathway (Cintas et al. 1997), and enterocin L50, which is secreted without a leader sequence (Cintas et al. 1998).

The storage stability of Japanese pickles is elevated by the effects of added salts and lactic acid produced by LAB. While lowering the salt content is beneficial for health, this undoubtedly leads the foods into a great risk of contamination. To compensate for the risk by lower salt concentration, LAB growing in the food are expected to increase the inhibition effect on contamination. More specifically, bacteriocin-producing LAB are hoped to be utilized as starter cultures that can inhibit the growth of other Gram-positive foodborne bacteria (Galvez et al. 2007). Nisin, the most typical bacteriocin, is widely used as a food preservative in more than 50 countries (Lubelski et al. 2008). In fact, nisin possesses wide and strong antimicrobial activity, but some bacteriocins are known to show higher activity against particular species than nisin as in the case of class IIa bacteriocins against Listeria. Given the application for fermented foods, if heterogeneous bacteriocin-producing LAB are used as starter cultures of fermented foods, they will inhibit LAB essential in the foods as well as other undesirable bacteria and will raise deterioration of the original taste. Isolates from the fermented foods themselves would be more appropriate antibacterial starters. These isolates may achieve not only the effective microbial control but also the enhancement of the taste retention and the shelf life of the foods. To utilize these types of LAB as starter cultures for certain fermented foods, we attempted to isolate bacteriocin-producing LAB from the fermented foods themselves. We isolated the strain QU 13 from a barrel to make Japanese traditional and regional pickle, Takanazuke. Strain QU 13 was shown to produce bacteriocins, which exhibited a unique antimicrobial spectrum. In this report, we described their purification and the structural and biological analyses.

Materials and methods

Bacterial strains and media

Strain QU 13, a bacteriocin-producing strain, was isolated from a wood barrel used to make pickle Takana (mustard greens), sampled in Fukuoka prefecture, Japan. Isolation sources, wood chips from the barrel, were enriched in MRS medium (Oxoid, Basingstoke, UK), and the culture was plated onto MRS agar plates containing 0·5% CaCO3. Then, LAB like colonies on these plates were isolated and tested for antimicrobial activity, and a bacteriocin-producing strain, termed strain QU 13, was selected for further study. Strain QU 13 was stored at −80°C in MRS medium with 15% glycerol and propagated in MRS broth at 30°C for 18 h before use. APT broth (BD, Sparks, MD, USA) was also used to culture strain QU 13. Indicator strains for the determination of antimicrobial activities were propagated at appropriate temperatures (30 or 37°C) for 18 h before use. LAB strains were grown in MRS medium, and the other Gram-positive indicator strains were grown in Tryptic Soy Broth (BD) supplemented with 0·6% yeast extract (Nacalai Tesque, Kyoto, Japan). Escherichia coli JM109 was grown in Luria-Bertani (LB) medium (BD), and transformants of E. coli JM109 were selected on LB agar plates containing 50 mg l−1 of isopropyl-β-d-thiogalactopyranoside and 20 mg l−1 of ampicillin.

Identification of strain QU 13

The sugar fermentation pattern of strain QU 13 was tested by the API 50 CHL system (bioMérieux, Marcy l’Etoile, France). The obtained pattern was analysed using the apiweb software (bioMérieux) and compared to the previous report (Collins et al. 1993). A partial 16S rDNA region of strain QU 13, corresponding to positions 8–1510 of E. coli 16S rDNA, was analysed as described previously (Zendo et al. 2005).

Determination of bacteriocin activity

Bacteriocin activity was determined by the spot-on-lawn method as described previously (Ennahar et al. 2001). Bacillus coagulans JCM 2257T was used as an indicator strain unless otherwise mentioned. Briefly, 10 μl of a bacteriocin preparation was spotted onto a double-layered agar plate with 5 ml of Lactobacilli Agar AOAC (BD) inoculated with an overnight culture of an indicator strain as the upper layer and 10 ml of MRS broth with 1·2% agar as the bottom layer. After overnight incubation at appropriate temperatures for the indicator strains, bacterial lawns were checked for inhibition zones. In the purification steps, the activity titre was defined as the reciprocal of the highest dilution that yielded a clear zone of growth inhibition in the indicator lawn; this value was expressed in arbitrary activity units (AU) per millilitre of bacteriocin preparation. Purified bacteriocin was diluted by sterile 0·1% (v/v) Tween 80 solution to appropriate concentrations and tested for antimicrobial activity against various indicator strains as described earlier. The minimum inhibitory concentration (MIC) was defined as the minimum bacteriocin concentrations that yielded clear zones of growth inhibition in the indicator lawn.

Purification procedure for bacteriocins

Bacteriocin purification was carried out by a three-step procedure using the supernatant of a 1-l culture of strain QU 13 grown for 20 h in APT broth at 30°C. The cell-free supernatant was mixed with 25 g of Amberlite XAD-16 (Sigma-Aldrich, St Louis, MO, USA), synthetic hydrophobic resin previously activated with 50% (v/v) isopropanol and equilibrated with distilled water. The mixture was gently shaken at 4°C overnight and transferred to a column (25 mm internal diameter, 300 mm length). After the mixture was washed with distilled water and 40% ethanol, bacteriocins were eluted with 200 ml of 70% isopropanol containing 0·1% trifluoroacetic acid (TFA). The active eluted solution was placed in a rotary evaporator (Tokyo Rikakikai, Tokyo, Japan) to remove isopropanol. The resulting solution was then diluted with 50 mmol l−1 sodium phosphate buffer (PB, pH 5·7) to 100 ml and loaded onto an SP-Sepharose Fast Flow cation-exchange column (15 mm internal diameter, 100 mm length; GE Healthcare, Uppsala, Sweden) pre-equilibrated with PB. The column was washed serially with 100 ml PB. Bacteriocins were eluted with 40 ml of PB containing 0·25 mol l−1 NaCl. For further purification, the active eluted solution was applied to a reversed-phase column (Resource RPC 3-ml; GE Healthcare) incorporated in the LC-2000Plus HPLC system (Jasco, Tokyo, Japan) and eluted with a gradient of MilliQ water–acetonitrile containing 0·1% TFA at a flow rate of 1 ml min−1 as follows: 0−10 min, 0%−40% (v/v); 10−50 min, 40−60% (v/v); and 50−60 min, 60−100% (v/v) acetonitrile. Active fractions were placed in a Speed-Vac Concentrator (Savants, Farmingdale, NY, USA) to thoroughly remove the acetonitrile. The purified bacteriocin solutions were used for MIC determination and further characterization such as amino acid sequence analysis and stability assessment as described later. The antimicrobial activity of each fraction in the purification steps was determined as described earlier. The protein concentration (mg ml−1) of each fraction was estimated from A280 by using a NanoDropR ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) according to the manufacturer’s instructions. Concentrations of purified bacteriocins were also calculated according to the following formula. These concentrations were used to determine the MICs.

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In these formulas, a, b and c are the respective number of Tyr, Trp and Cys residues per molecule, where εtyr is 1280 M−1 cm−1, εtrp is 5690 M−1 cm−1 and εcys is 120 M−1 cm−1.

Mass spectrometry and amino acid sequencing

Molecular mass analyses of purified bacteriocins were conducted by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) with a JMS-T100LC mass spectrometer (JEOL, Tokyo, Japan). The N-terminal amino acid sequences of the purified bacteriocins were determined by Edman degradation using a PPSQ-21 gas-phase automatic protein sequencer (Shimadzu, Kyoto, Japan). Deformylation treatment with 0·6 mol l−1 HCl and fragmentation with BNPS-skatole or CNBr were performed as previously described to access the N-terminals and obtain further amino acid sequences (Sawa et al. 2009).

Effects of pH and temperature on bacteriocin stability

To evaluate the heat and pH stability, the purified bacteriocins were adjusted to pHs between 3·0 and 11·0 using 1 mol l−1 HCl or 1 mol l−1 NaOH. The preparations were then heated at 80, 100 or 121°C for 30 min. The preparations were also left at room temperature (25°C) overnight. The residual activities were determined as described earlier. The activity of the samples of pH 3·0 without heat treatment was considered as 100%.

Analysis of the genes encoding the bacteriocins

DNA manipulations were performed according to the previous report (Fujita et al. 2007). DNA polymerases, restriction enzymes and other DNA-modifying enzymes were used according to the manufacturer’s instructions. Total DNA of strain QU 13 was extracted from cells treated with lysozyme (Seikagaku, Tokyo, Japan) and cetyltrimethylammonium bromide (Wako, Osaka, Japan), according to procedures described previously (Fujita et al. 2007), and used as a template for PCR and subsequent procedures. The oligonucleotide primers used in this study are listed in Table 1. Degenerate primers, pAD1, pAD2, pBD1, and pBD2, designed from the obtained amino acid sequences of the bacteriocins were used to amplify a fragment corresponding to a part of the genes encoding the bacteriocins. Degenerate PCR was performed using Taq DNA polymerase (Promega, Madison, WI, USA) under the following conditions: denaturation at 94°C for 3 min; 30 cycles of denaturation at 94°C for 30 s, annealing at optimal temperature of each primer for 30 s and polymerization at 72°C for 15 s. The PCR fragment was cloned into E. coli JM109 by using pGEM-T vector systems (Promega). DNA sequencing was carried out by Macrogen Inc. (Seoul, Korea). To obtain the upstream and downstream sequences of bacteriocins’ structural genes, inverse PCR was performed. Total genomic DNA of strain QU 13 was completely digested with MboI or EcoRI, and self-ligated for use as templates. Inverse PCR was performed with KOD plus DNA polymerase (Toyobo, Osaka, Japan) using primers pA1, pA2, pB1 and pB2 designed from the sequence obtained by the degenerate PCR, under the following conditions: denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at optimal temperature of each primer for 30 s and polymerization at 68°C for 2·5 min. The products obtained by inverse PCR were cloned and sequenced as described earlier. On the basis of sequences obtained from the inverse PCR products, new specific primers, wY3f, wY8r, wM1f and wM9r, were synthesized and used to confirm the obtained DNA sequence, according to the procedures described earlier.

Table 1.   Oligonucleotide primers used to obtain the biosynthesis gene cluster of weissellicins Y and M
Primer nameSequence (5′–3′)
pAD1ATGGCNAAYATHGTNYTNMGN
pAD2CCAYTTDATDATYTGRTTRTA
pBD1ATGGTNWSNGCNGCNAARGT
pBD2CCAYTCNGCDATYTGRTCNAC
pA1GGCGCATAATTATATGCAACAGA
pA2GAGTTTCTTACAACCAGATCATT
pB1CTAACCCCCAACCAACTTTTAAA
pB2AATTTATTGGTGAGGGTTGGTCT
1stMup13-fTTAACTATGCGGCATCAGA
1stMup13-rTAATGTGAGTTAGCTCACTC
Mup13-fAAGGCGATTAAGTTGGGTA
Mup13-rGTATGTTGTGTGGAATTGTG
s-M13-fGTAAAACGACGGCCAGT
s-M13-rTTCACACAGGAAACAGG
wY3fTATGCCAGCTTAGATCATGTAACT
wY8rGATTTGAACGACGAAATTAGTTGTTC
wM1fTTATAAATATTAAAAACAAGCCT
wM9rGGTAGTTTACATCTGACAACTGAC

Computer analysis of DNA and amino acid sequences

The obtained DNA and amino acid sequences were analysed using Genetyx-Win software ver. 8.0.1 (Genetyx, Tokyo, Japan). Database searches were performed using the Blast program of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/BLAST/).

DNA sequence accession number

The nucleotide sequence determined in this study has been deposited in the DDBJ database under accession no. AB501211 and AB670118.

Results

Identification of strain QU 13

Using API 50 CHL system, the sugar fermentation pattern test of strain QU 13 was carried out, and strain QU 13 could not be assigned to a particular genus (data not shown). The 16S rDNA sequence of strain QU 13 showed 99% identity to those of the reference strains of Weissella hellenica and Weissella paramesenteroides. Other characteristics of strain QU 13 such as Gram-positive and hetero l-lactic acid production from glucose also agreed well with the characteristics of both the species (data not shown). The presence of characteristic signatures in the 16S rDNA is known to provide further evidence for identification. The most notable signature for Weissella is the diagnostic sequences in 1007/1022 of the variable region V6, which readily distinguishes an unknown bacterium from all known species of the Weissella genus (Collins et al. 1993). In this region, strain QU 13 showed 100% homology with W. hellenica but proved to be significantly different from W. paramesenteroides (data not shown). Considering these results, we concluded that strain QU 13 belonged to W. hellenica.

Purification of bacteriocins produced by strain QU 13

The bacteriocins produced by strain QU 13 were purified by a three-step procedure, which included hydrophobic interaction, cation-exchange chromatography and reversed-phase HPLC. Most of the activity in the culture supernatant was recovered by hydrophobic interaction chromatography. In cation-exchange chromatography, the bacteriocin activity was mainly recovered in 0·25 mol l−1 NaCl fraction. This fraction was applied subsequently to reversed-phase HPLC. Two peaks (peptides A and B) with antimicrobial activity were obtained (Fig. 1). Finally, we could obtain approximately 53% activity of the total activity of the culture supernatant by these purification steps, the details of which are summarized in Table 2.

Figure 1.

 Reversed-phase HPLC of bacteriocins from Weissella hellenica QU 13. The bacteriocin activity was detected in the fractions containing the peaks indicated by A and B. The broken line indicates the acetonitrile gradient.

Table 2.   Purification of weissellicins Y and M produced by Weissella hellenica QU 13
StepsVolume (ml)Total act. (AU)*Yield (%)Total protein (mg)Purification (fold)
  1. *Antimicrobial activity [in arbitrary units (AU)] was assayed by the spot-on-lawn method using Bacillus coagulans JCM 2257T as an indicator strain.

Supernatant10004·00 × 1051002·27 × 1041·00
Amberlite XAD-162006·40 × 1051602·40 × 10315·1
SP-Sepharose405·12 × 1051286·20 × 1046·9 × 10
RP-HPLC
 Weissellicin Y16·00 × 1031·50·39093·0 × 10
 Weissellicin M12·05 × 10551·30·89013·0 × 103

Mass spectrometry and amino acid sequence analysis

ESI-TOF MS analysis showed that purified peptides A and B had molecular masses of 4925·12 and 4967·30 Da, respectively (Fig. 2). Both molecular masses were not identical to those of any known bacteriocins. Therefore, we concluded that peptides A and B were novel bacteriocins and designated them weissellicin Y and weissellicin M, respectively. Edman degradation of weissellicins Y and M did not proceed, but after deformylation treatment with 0·6 mol l−1 HCl, the degradation was accessible. This indicated that N-terminal residues of both peptides were blocked by formylation. With BNPS-skatole treatment or CNBr treatment before Edman degradation, each bacteriocin was fragmentized, and the resulting fragments were fractionated by HPLC. These fragments were sequenced by Edman degradation. Consequently, the sequencing analyses revealed the N-terminal sequences of 35 amino acid residues (MANIVLRVGSVAYNYAPKIFKWIGEGVSYNQIIKW) of weissellicin Y and 28 amino acid residues (MVSAAKVALKVGWGLVKKYYTKVMQFIG) of weissellicin M. The respective calculated masses of 4099 and 3051 Da, including N-terminal formyl groups (+28 Da), were not enough to their molecular masses observed by ESI-TOF MS. This implies that the obtained amino acid sequences of weissellicins Y and M were not complete.

Figure 2.

 Electrospray ionization time-of-flight mass spectra of purified weissellicins Y and M. Weissellicins Y (a) and M (b) correspond to peaks A and B in Fig. 1, respectively. Multiple charged molecular ions were detected and are indicated. Molecular masses were calculated from the values of the major peaks of tetravalent or pentavalent ions.

Antimicrobial spectra of weissellicins Y and M

Purified bacteriocins were tested for antimicrobial activity against various indicator strains by the spot-on-lawn method (Table 3). Weissellicin Y showed relatively weak activity against almost all the indicator strains, and comparatively high activity against B. coagulans, which is considered to be a major contaminant of pickles. Furthermore, weissellicin Y showed higher antimicrobial activity against closely related bacteria, namely, Weissella spp. On the other hand, weissellicin M indicated a very broad antimicrobial spectrum and showed remarkably high activity against B. coagulans, Bacillus subtilis, Lactococcus lactis, Enterococcus faecium, Streptococcus bovis and also Weissella spp. Overall, weissellicin M showed much higher activity than weissellicin Y, but their activities against Leuconostoc mesenteroides were almost at the same level. In addition, against the nisin producer L. lactis JCM 7638 (Matsusaki et al. 1998), weissellicin M showed significant activity, while weissellicin Y showed no activity even at the concentration of 52·2 μmol l−1. Weissellicins Y and M did not show any synergistic activity (data not shown).

Table 3.   Antimicrobial spectra of weissellicins Y and M
Indicator strainsMICs (μmol l−1)
Weissellicin YWeissellicin M
  1. ATCC, American Type Culture Collection, Rockville, MD, USA; JCM, Japan Collection of Microorganisms, Saitama, Japan; NBRC, NITE Biological Resource Center, Chiba, Japan; MIC, minimum inhibitory concentration; N.A., no activity even by maximum concentrations of weissellicin Y and weissellicin M, 52·2 and 13·5 μmol l−1, respectively.

Lactococcus lactis ssp. lactis JCM 7638N.A.1·41
L. lactis ssp. lactis ATCC 19435T1·630·0440
Llactis ssp. lactis NCDO 4971·630·0440
Lactobacillus sakei ssp. sakei JCM 1157T3·260·354
Lactobacillus plantarum JCM 1149T13·10·176
Weissella cibaria JCM 12495T1·630·0440
Weissella hellenica JCM 10103T0·4080·0220
Weissella paramesenteroides JCM 9890T0·8160·0220
Weissella confusa JCM 1093T0·4080·0440
Pediococcus pentosaceus JCM 58856·532·83
Pediococcus dextrinicus JCM 5887T13·12·83
Pediococcus acidilactici JCM 8797T13·11·41
Enterococcus faecium JCM 5804T6·530·0880
Enterococcus durans NBRC 100479T13·10·176
Enterococcus faecalis JCM 5803T52·22·83
Streptococcus salivarius JCM 5707TN.A.N.A.
Streptococcus bovis JCM 5802T3·260·0880
Streptococcus mutans JCM 5705TN.A.N.A.
Streptococcus dysgalactiae ssp. dysgalactiae JCM 56733·260·176
Bacillus coagulans JCM 2257T0·4080·0220
Bacillus circulans JCM 2504T13·10·354
Bacillus subtilis ssp. subtilis JCM 1465T3·260·0880
Bacillus cereus JCM 2152T6·531·41
Kocuria rhizophila NBRC 1270813·10·708
Listeria innocua ATCC 33090T26·12·83
Leuconostoc mesenteroides ssp. mes. JCM 6124T3·261·41
Escherichia coli JM109N.A.N.A.
Ecoli NBRC 3301N.A.N.A.
Staphylococcus aureus ssp. aureus ATCC 12600TN.A.N.A.

Effects of pH and temperature on bacteriocin stability

The antimicrobial activities of weissellicins Y and M were assayed under a wide range of pH and temperature conditions (Fig. 3). The activity of weissellicin Y diminished upon exposure to elevated temperatures and high pH (Fig. 3a). Under autoclaving conditions at 121°C for 30 min, the activity of weissellicin Y was almost inactivated, whereas at least 25% of the initial activity was retained at 80°C. In contrast, weissellicin M showed very high stability against high temperatures and pH (Fig. 3b). Even at 121°C and pH 3, it still retained 100% of its initial activity. Weissellicin M also retained 50% of its activity after exposure to alkaline pH range at room temperature overnight and at 80°C for 30 min.

Figure 3.

 Effects of pH and temperature on the activities of weissellicins Y and M. Purified bacteriocins (weissellicins Y (a) and M (b)) adjusted to a pH range of 3·0–11·0 were incubated at 80, 100 or 121°C for 30 min. In addition, each pH-treatment sample was incubated at 25°C overnight (o/n). The activity of the samples at pH 3·0 without any treatment was considered to be 100%. The colours of bars indicate pH 3·0 (black), 5·0 (dark grey), 7·0 (grey), 9·0 (light grey) and 11·0 (white) treatments.

Analysis of the gene encoding weissellicins Y and M

The structural gene sequences of weissellicins Y and M were obtained by degenerate PCR and subsequent inverse PCR by using primers shown in Table 1. Sequencing analysis revealed two orfs, termed welY and welM, encoding weissellicins Y and M, respectively, and consisting of respective 42 and 43 amino acid residues (Fig. 4). The calculated masses of 4925 and 4967 Da, including formyl groups (+28), agreed completely with those observed by ESI-TOF MS. This indicated that all the amino acid sequences of weissellicins Y and M were completely elucidated. This also suggested that both weissellicins Y and M were transcribed with neither an N-terminal leader sequence nor an N-terminal signal peptide, which is different from most bacteriocins that are generally transcribed with a leader peptide (Nes et al. 1996).

Figure 4.

 Nucleotide sequences of the regions encoding weissellicins Y (a) and M (b), as well as the deduced amino acid sequences. The putative ribosome-binding sites (RBS) are underlined, and asterisk means the stop codon.

The database search showed that weissellicin Y was a novel bacteriocin with no homology with any known bacteriocins and that weissellicin M was also a novel bacteriocin with significant identity with enterocins L50A and L50B, which are produced by Ent. faecium L50 (Cintas et al. 1998). The region around all the methionine and tryptophan residues and the C-terminal region of weissellicin M showed distinctively high identity with those of enterocins L50A and L50B (Fig. 5).

Figure 5.

 Alignment of weissellicin Y, weissellicin M and enterocins L50A and L50B. Identical amino acid residues are indicated by grey boxes.

Further DNA sequence analyses with inverse PCRs revealed additional DNA sequences around each structural gene, indicating the structural genes are located in the same locus with the distance of around 3 kb (Fig. 6a). Among the six putative orfs obtained, the protein sequences translated from three orfs (orf2, orf3 and orf5) showed significant identities with those from the respective putative genes involved in biosynthetic gene clusters of enterocins L50A and L50B, p10 (hypothetical protein, 26%), L50G (putative transporter, 27%) and L50E (putative transporter, 40%) (Fig. 6b). orf1, orf4 and orf6 did not show significant identity to any known function proteins.

Figure 6.

 Putative biosynthesis gene clusters of weissellicins Y and M (a) and enterocin L50 (Criado et al. 2006a) (b). White arrows denote respective similar orfs (orf5-L50E, orf3-L50G and orf2-p10).

Discussion

A few preliminary studies on bacteriocins from Weissella genus have been previously presented (Papathanasopoulos et al. 1997; Srionnual et al. 2007; Papagianni and Papamichael 2011). To our knowledge, the present study is the first report that clarifies the structural genes as well as complete primary structures of bacteriocins from Weissella genus.

Weissella hellenica QU 13 was isolated from a Japanese pickles barrel, and two novel bacteriocins, namely weissellicins Y and M, were purified from the culture supernatant of W. hellenica QU 13. Both the N-terminal methionines were blocked by formylation, and both the bacteriocins were synthesized without N-terminal leader sequences or signal peptides. Most LAB bacteriocins were synthesized as prepeptides containing N-terminal extension or leader sequences, while some class II bacteriocins have been reported to be synthesized without a leader sequence. The latter includes mutacin BHT-B (Hyink et al. 2005), lacticin Q (Fujita et al. 2007), lacticin Z (Iwatani et al. 2007), enterocin Q (Cintas et al. 2000) and enterocin L50 (Cintas et al. 1998; Criado et al. 2006a; Izquierdo et al. 2008), most of which are class IId bacteriocins.

Because the peaks of the bacteriocins in final purification step of HPLC were close as shown in Fig. 1, contaminations of each peptide were carefully checked. Accordingly, by ESI/MS analysis, no contamination of another bacteriocin was observed in each purified fraction (Fig. 2). In addition, heterologous expressions that produced weissellicin Y or weissellicin M singly were performed. From the culture of each transformant, respective bacteriocins were purified and both showed significant activities (data not shown). In fact, antimicrobial spectra and stabilities of purified weissellicin Y and weissellicin M were quite different.

The antimicrobial spectra of weissellicins Y and M were evaluated by the spot-on-lawn method against various indicator strains, and MICs were determined (Table 3). Weissellicin M showed a broad antimicrobial spectrum and distinctively high activity. Thus, weissellicin M has significant potential to be utilized for pickles preservation. The antimicrobial activity of weissellicin Y was totally weaker than that of weissellicin M; however, the only exception was the case of the indicator strain Leuc. mesenteroides, against which both weissellicins Y and M showed almost same activity. In addition, against L. lactis JCM 7638, their activities were quite different. Weissellicin M showed significant antimicrobial activity against the nisin producer strain L. lactis JCM 7638 (Matsusaki et al. 1998), while weissellicin Y showed no activity. Consequently, weissellicins Y and M were proved to have different antimicrobial spectra.

Many bacteriocins, including nisin, are easily inactivated by alkali pH treatment (Delves-Broughton et al. 1996). As in the cases of most of other bacteriocins, weissellicin Y was inactivated by alkaline pH and high temperature, while weissellicin M showed higher stability against pH and heat (Fig. 3). After overnight pH treatments, weissellicin M retained more than 50% of its initial antimicrobial activity. Furthermore, in the acidic pH range, even after autoclaving at 121°C, weissellicin M retained more than 50% activity. Weissellicin M can be used under broad heat and pH conditions, which makes it useful for many industrial applications.

The amino acid sequence homology analysis revealed that weissellicin Y showed no significant homology with any other bacteriocins, while weissellicin M showed very high homology with enterocin L50, which is produced by Ent. faecium L50 that also produces enterocins P and Q (Criado et al. 2006a,b; Basanta et al. 2008). Enterocin L50 consists of two peptides, namely, enterocin L50A and L50B, and both are synthesized without leader sequences or signal peptides. In addition, their N-terminal methionines are formylated. As in the case of enterocin L50, weissellicins Y and M bore no N-terminal extensions, and N-terminal methionines of both bacteriocins were formylated. However, only weissellicin M showed high identity with both enterocins L50A and L50B (54 and 59%, respectively), particularly at the regions around all methionine and tryptophan residues, and at the C-terminal region (Fig. 5). Indeed, weissellicin M appears a hybrid of enterocins L50A and L50B.

Some previous studies have reported about multiple-bacteriocin-producing LAB such as Lactobacillus (Vaughan et al. 2003), Enterococcus (Cintas et al. 1998; Izquierdo et al. 2008) and Leuconostoc (Papathanasopoulos et al. 1997). Enterococcus faecium L50 produces four different bacteriocin peptides, enterocins L50A, L50B, P and Q, and controls the production of the bacteriocins depending on culture temperature (Cintas et al. 2000; Criado et al. 2006b). Orf2, 4 and 5 in W. hellenica QU 13 showed high similarity with putative biosynthetic genes of enterocin L50, and the structural genes welY and welM appeared to belong to different operons, which were neither adjacent to each other nor in the same orientation. In fact, it is still not sure whether weissellicin Y and weissellicin M are produced by same biosynthesis machinery. These suggest, however, that W. hellenica QU 13 can independently regulate the transcriptions of welY and welM responding to alterations in the environments. The regulation property of weissellicins Y and M production with various conditions will be elucidated by quantification analysis of the peptides and transcriptional analysis, targeting not only the structure genes but also the related biosynthetic genes.

Weissella hellenica QU 13 and Ent. faecium L50 share some very unique characteristics such as production of multiple-bacteriocins, class IId bacteriocin production with no N-terminal extension and N-terminal methionine blockage because of formylation. These unique characteristics of both strains make them extremely important from a biological perspective. For example, both strains would have novel bacteriocin secretion systems, which are different from other known bacteriocin systems. Both strains may also regulate bacteriocin production while adapting to environmental conditions. Upon the clarification of their regulatory mechanisms, it will be possible to control production of each bacteriocin for various purposes or target bacteria from a single bacteriocin-producing strain.

From the view point of evolution, environmental biology and antibacterial starter culture, the multiple-bacteriocin-producing LAB, W. hellenica QU, 13 is of great importance. In our future work, the weissellicins Y and M biosynthetic system, from transcription to secretion and immunity, will be clarified. Furthermore, studies on its application will be expected.

Acknowledgements

This work was partially supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), the Research Grant for Young Investigators of Faculty of Agriculture, Kyushu University, and Kato Memorial Bioscience Foundation.

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