Weissellicin L, a novel bacteriocin from sian-sianzih-isolated Weissella hellenica 4-7

Authors


Correspondence

Yi-Sheng Chen, Department of Biotechnology, Ming Chuan University, No. 5, De-Ming Rd., Gui-Shan Township, Taoyuan County 333, Taiwan.E-mail: yisheng@mail.mcu.edu.tw

Abstract

Aim

To identify and characterize novel bacteriocins from Weissella hellenica 4-7.

Methods and Results

Weissella hellenica 4-7, isolated from the traditional Taiwanese fermented food sian-sianzih (fermented clams), was previously found to produce a bacteriocin active against Listeria monocytogenes and some other Gram-positive bacteria. Bacteriocin activity decreased slightly after autoclaving (121°C for 15 min), but was inactivated by protease K and trypsin. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 3205·6 Da. N-terminal amino acid sequencing yielded a partial sequence, NH2-KGFLSWASKATSWLVGP, by Edman degradation. The obtained partial sequence showed high homology with leucocin B-TA33a; however, at least two different residues were observed. No identical peptide or protein was found, and this peptide was therefore considered to be a novel bacteriocin produced by W. hellenica 4-7 and termed weissellicin L.

Conclusions

The findings obtained in the current study suggest a novel bacteriocin produced by W. hellenica 4-7.

Significance and Impact of the Study

Bacteriocins from Weissella remain rare, and this study is the second report of a bacteriocin produced by W. hellenica.

Introduction

Lactic acid bacteria (LAB) play an important role in food fermentation and contribute to the organoleptic properties of the final product, as well as its preservation and microbial safety. Antimicrobial activities of LAB, such as the production of lactic acid and ethanol, could also be associated with ribosomally synthesized proteinaceous inhibitors collectively known as ‘bacteriocins’. Bacteriocins are of great interest to the food industry as natural preservatives and possible substitutes for chemical preservation (Cleveland et al. 2001; Ross et al. 2002). Bacteriocins are peptides produced by bacteria that kill or inhibit the growth of closely related bacteria.

Bacteriocins from Lactobacillus, Enterococcus, Lactococcus and Leuconostoc have been frequently reported (Casaus et al. 1997; Ennahar et al. 1999; Foulquié Moreno et al. 2003; Zendo et al. 2003; Settanni and Corsetti 2008; Masuda et al. 2011; Chahad et al. 2012; Nishie et al. 2012; Lin et al. 2013). Several bacteriocins from Weissella, such as weissellicin 110, weissellin A, weissellicin Y and weissellicin M, have been previously reported (Srionnual et al. 2007; Papagianni and Papamichael 2011; Masuda et al. 2012). With the exception of weissellicin Y and weissellicin M, reported by Masuda et al. (2012), the study of bacteriocins from Weissella hellenica remains rare.

In a previous study, we reported that W. hellenica 4-7, isolated from fermented clams (sian-sianzih), is capable of producing a bacteriocin-like inhibitory substance (BLIS) (Chen et al. 2012). However, the primary structure and biochemical characteristics of the BLIS from W. hellenica 4-7 have not yet been discussed. The current study describes the purification and analysis of a bacteriocin from strain W. hellenica 4-7 and discusses its similarities to other known peptides.

Materials and methods

Bacterial strains used in this study

Weissella hellenica 4-7 was grown in a modified glucose yeast peptone (GYP) medium containing 2·0% dextrose, 1·5% polypeptone, 0·5% yeast extract, 0·5% sodium acetate and 0·3% (v/v) salt solution (4% MgSO4・7H2O, 0·16% MnSO4・4H2O, 0·2% FeSO4・7H2O and 0·2% NaCl), pH 6·8, at 30°C under aerobic condition without shaking (Chen et al. 2007). Weissella paramesenteroides ATCC 33313T was used as the indicator strain for inhibitory activity assay and was grown in de Man Rogosa and Sharpe (MRS) (De Man et al. 1960) medium (BD, Franklin Lakes, NJ, USA) at 30°C. Inhibitory activity was determined using the agar-well diffusion assay. Briefly, a 2 ml of soft agar (0·5% w/v) was seeded with 100 μl of an overnight culture of the indicator strain and used to overlay an agar plate. Antibacterial activity was determined by pouring the supernatant fluid into the hole made on the agar plates. Plates were incubated overnight at 30 or 37°C and examined for clear zones of inhibition (Table 1).

Table 1. Characteristics of the bacteriocin from Weissella hellenica 4-7
 Diameter of inhibition zone (mm)a,b
  1. a

    Determined using the agar-spot test. Crude bacteriocin treatments (2 μl) were spotted.

  2. b

    Average value of duplicate tests.

Growth temperature (°C)
 2511
 3012
 3710
 450
Enzymes
 Control11
 Proteinase K0
 Trypsin0
 Catalase11
Heat
 Control11
 30 min at 80°C11
 30 min at 90°C11
 15 min at 121°C10

Optimum temperature for bacteriocin production

To determine the optimum temperature for bacteriocin production, 100 μl of overnight bacterial culture was inoculated into 5 ml MRS broth and then incubated at 25, 30, 37 or 45°C for 24 h. After incubation, bacteriocin activity was determined using the size of the inhibition zone in the agar-well diffusion assay. W. paramesenteroides ATCC 33313T was used as the indicator strain.

Production of bacteriocin by Weissella hellenica 4-7

To study bacteriocin production, 10 ml of overnight bacterial culture was inoculated into 990 ml of GYP medium. At specific time intervals, 1-ml samples were removed, and the optical density at 600 nm of the culture and the arbitrary activity units (AU) ml−1 (reciprocal of the highest dilution at which activity was still obtained) of the bacteriocin were determined according to the method of Srionnual et al. (2007). W. paramesenteroides ATCC 33313T was used as the indicator strain. The incubation temperature was set based on the results obtained from the optimum growth temperature analysis.

Preparation of crude bacteriocin sample

Crude bacteriocin was initially obtained and purified by pH-dependent adsorption and desorption method onto producer cells (Yang et al. 1992). Briefly, W. hellenica 4-7 was grown in 0·8 l modified GYP medium at 30°C for 16 h. The pH was adjusted to 6·0, and the culture pellet was harvested by centrifugation for 20 min at 6100 g. The culture pellet was washed twice in 5 mmol l−1 sodium phosphate buffer (pH 6·0) and resuspended in 40 ml of 0·1 mol l−1 NaCl solution adjusted to pH 2·0 with 5% (v/v) phosphoric acid. The suspension was stirred for 1 h, and cells were removed by centrifugation at 12 400 g for 20 min. The supernatant was filtered through a 0·45-μm-pore-size membrane (Advantec, Tokyo, Japan) and then desalted using a C18 cartridge (Sep-Pak C18, Waters, Milford, MA, USA), prewashed with acetonitrile containing 0·1% trifluoroacetic acid (TFA) and conditioned with 0·1% TFA. The crude bacteriocin was eluted in 30% acetonitrile with 0·1% TFA (Yanagida et al. 2005). Eluted bacteriocin fractions were freeze-dried and stored at −20°C until further study.

Effects of enzymes and heat on bacteriocin activity

Before tests, the crude bacteriocin powder was weighed and then thousand-fold (w/v) diluted by resuspending in 30% acetonitrile solution. To evaluate heat stability, the crude bacteriocin solution was incubated at 80°C for 30 min, 90°C for 30 min or 121°C for 15 min. To analyse sensitivity to various enzymes, the crude bacteriocin solution was treated with proteinase K (Merck, Darmstadt, Germany), trypsin (Wako, Osaka, Japan) or catalase (Wako) (30 IU mg−1, 37°C for 5 h). Next, the inhibitory activity of individual 2 μl crude bacteriocin treatments was determined using the agar-spot test (Schillinger and Lücke 1989). Briefly, a 2 ml of soft agar (0·5% w/v) was seeded with 100 μl of an overnight culture of W. paramesenteroides ATCC 33313T and used to overlay MRS plate. Antibacterial activity was determined by spotting 2 μl of each crude bacteriocin treatment onto the surface of MRS agar plate. Plate was incubated overnight at 30°C, and the size of clear zone was determined.

Inhibition spectrum of bacteriocin

The purified bacteriocin solution was further tested against the Gram-positive and Gram-negative bacteria listed in Table 2. The inhibitory activity assay was performed using the agar-spot test method.

Table 2. Inhibition spectra comparison of weissellicin L and other known bacteriocins from Weissella
Indicator strainsMediumIncubation temperature (°C)Weissellicin LbWeissellicin 110bWeissellicin YcWeissellicin McWeissellin AdOrigin
  1. +, inhibitory zone observed; −, no inhibitory zone observed; NA, data not available.

  2. a

    Type strain obtained from BCRC (The Bioresource Collection and Research Center).

  3. b

    Data obtained from the present study.

  4. c

    Data obtained from study of Masuda et al. (2012).

  5. d

    Data obtained from study of Papagianni and Papamichael (2011).

Listeria monocytogenes ATCC 19111BHI37+NANA+ a
Listeria innocua ATCC 33090TTSB37NANA++NAMasuda et al. (2012)
Lactobacillus sakei subsp. sakei JCM 1157TMRS30+++++ a
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842TMRS37+NANANA a
Weissella paramesenteroides ATCC 33313TMRS30++++NA a
Weissella hellenica ATCC 51523TMRS30++++NA a
Weissella minor ATCC 35412TMRS30NANANA a
Weissella viridescens ATCC 12706TMRS30+NANANA a
Lactococcus lactis subsp. lactis ATCC 19435TMRS30+++NA a
L. lactis subsp. cremoris ATCC 19257TMRS30+NANANA a
L. lactis subsp. hordniae ATCC 29071TMRS30+NANANA a
L. lactis subsp. tructae DSM 21502TMRS30NANANA a
Lactococcus taiwanensis 0905C15TMRS30+NANANA a
Lactococcus garvieae E-9MRS30NANANALin et al. (2013)
L. garvieae E-10MRS30NANANALin et al. (2013)
Streptococcus thermophilus ATCC 19258TMRS37++NANANA a
Escherichia coli ATCC 11775TLB37NA a
Vibrio vulnificus ATCC 27562TBHI37NANANA a
Bacillus subtilis subsp. subtilis ATCC 6051TLB37++NA a

Molecular size approximation

The molecular size of the crude bacteriocin was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), following the method described by Yanagida et al. (2005). A 4–20% Mini-Protean TGX precast gel (Bio-Rad Laboratories, Hercules, CA, USA) was used, and BLIS molecular size was estimated using Precision Plus Protein Dual Xtea Standards (Bio-Rad). W. paramesenteroides ATCC 33313T was used as the indicator strain.

Purification of the bacteriocin

An approximately 2 mg freeze-dried crude bacteriocin sample was resuspended in 0·2 ml of 30% acetonitrile containing 0·1% TFA. The crude bacteriocin solution was subsequently placed on a C18 reverse-phase column (BioBasic-18; Thermo Scientific, Rockford, IL, USA) integrated into a high-performance liquid chromatography (HPLC) system (L-2130, L-2450; Hitachi, Tokyo, Japan). The bacteriocin was initially eluted with a gradient of MilliQ water–acetonitrile containing 0·1% TFA, at a flow rate of 1·0 ml min−1, as follows: 0–10 min, 10–20% (v/v); 10–65 min, 20–100% (v/v); and 65–70 min, 100% (v/v) acetonitrile. The bacteriocin activity of fractions obtained from the first purification procedure was determined against W. paramesenteroides ATCC 33313T. The active eluted solution was freeze-dried on an EYELA FDU-2200 freeze dryer (Tokyo Rikakikai, Tokyo, Japan). The active eluted sample was resuspended and then re-integrated into the HPLC system using the same C18 reverse-phase column (BioBasic-18; Thermo Scientific, Waltham, MA, USA). The bacteriocin was eluted with a gradient of MilliQ water–acetonitrile containing 0·1% TFA, at a flow rate of 0·9 ml min−1, as follows: 0–10 min, 30–45% (v/v); and 10–60 min, 45–70% (v/v) acetonitrile. Detection of peptides was monitored using a UV detector at 220 nm. Antimicrobial activity of detected peaks was tested using the agar-spot diffusion assay.

Mass spectrometry

The molecular mass of the purified bacteriocin was determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) using a mass spectrometer (SCIEX QSTAR Elite; Applied Biosystems, Foster City, CA, USA).

N-terminal amino acid sequence analyses

The active peak obtained from the HPLC analysis was further subjected to N-terminal amino acid sequencing using an ABI Procise 494 protein sequencer (Applied Biosystems).

Results

Maximum cell numbers and inhibitory activity against the indicator strain W. paramesenteroides ATCC 33313T were observed at 30°C (Table 1). The highest bacteriocin titres (160 AU ml−1) were obtained after 22 h of incubation at 30°C, and the highest cell density, based on OD at 600 nm, was observed after 18 h of incubation (Fig. 1). Duplicate tests were performed, and similar results were obtained.

Figure 1.

Production of bacteriocin during the growth of Weissella hellenica 4-7. Symbols: green circle, cell number at OD 600; blue triangle, pH value; red bar, arbitrary activity (AU ml−1).

The effects of enzymes and heat on the inhibitory agent from Weissella hellenica 4-7 are shown in Table 1. The crude bacteriocin was inactivated by proteinase K or trypsin, but was not affected by catalase treatment. The bacteriocin was considered to be heat stable, as the majority of activity remained after heating at 121°C for 15 min (Table 1).

The purified bacteriocin solution was tested against some Gram-positive and Gram-negative bacteria. The purified bacteriocin solution of W. hellenica 4-7 showed inhibitory activity against Listeria monocytogenes ATCC 19111 and some Gram-positive bacteria, as listed in Table 2, but had no activity against Escherichia coli ATCC 11775T and Vibrio vulnificus ATCC 27562T.

The molecular size of crude bacteriocin from W. hellenica 4-7 was analysed by SDS-PAGE, and the specific band associated with the antibacterial activity was approximately 5–10 kDa (Fig. 2).

Figure 2.

SDS-PAGE analysis of the crude bacteriocin from Weissella hellenica 4-7. Left side: CBB-stained gel and right side: gel placed onto MRS agar surface overlaid with Weissella paramesenteroides ATCC 33313T. Lane M, Precision Plus Protein Dual Xtea Standards (Bio-Rad); lanes 1 and 2, crude bacteriocin from W. hellenica 4-7.

In the first purification step of HPLC, a peak with bacteriocin activity was observed at 39·1 min (Fig. 3a). A fraction of this peak was collected and used for the second purification procedure. A peak with bacteriocin activity was observed at 24·3 min in the second purification step (Fig. 3b). This peak was therefore collected and used for further study.

Figure 3.

The C18 reverse-phase chromatography profile of the bacteriocin from Weissella hellenica 4-7. (a) The first purification procedure: the peak with bacteriocin activity was observed at 39·1 min, indicated by black arrow at top. (b) The second purification procedure: the peak with bacteriocin activity was observed at 24·3 min, indicated by black arrow at top.

The purified bacteriocin was further analysed by MALDI-TOF-MS, and the result showed a major peak at 3205·64 Da (Fig. 4). N-terminal amino acid analysis of the purified bacteriocin revealed the following partial sequence: NH2-KGFLSWASKATSWLVGP.

Figure 4.

MALDI-TOF mass spectrum analysis of the purified bacteriocin from Weissella hellenica 4-7 (weissellicin L) shows the mass (m/z) of 3205·64 Da.

A similar sequence (Accession No.: P81052) was found in the DDBJ database (http://blast.ddbj.nig.ac.jp/blast/blastp?lang=en). It was marked as leucocin B-TA33a from Leuconostoc mesenteroides TA33a. When comparing the bacteriocin sequences, two different amino acid residues were observed between the bacteriocin from W. hellenica 4-7 and leucocin B-TA33a (Fig. 5). In addition, leucocin B-TA33a is the only sequence that shows similarity with our bacteriocin; there is no other sequence with significant similarity found in the database. The results described above strongly suggest that W. hellenica 4-7 naturally produces a novel bacteriocin, which we have termed weissellicin L.

Figure 5.

Multiple alignment of weissellicin L with leucocin B-TA33a, weissellicin 110, weissellin A, weissellicin Y and weissellicin M. Amino acid residues that are the same with those of weissellicin L are highlighted in grey.

Discussion

Growth temperature has an influence on the production of bacteriocins (Drosinos et al. 2005; Van den Berghe et al. 2006). The results obtained in the current study indicated that Weissella hellenica 4-7 had higher bacteriocin production when incubated at 30°C for 24 h than when incubated at other temperatures (Table 1). The sensitivity of the substance to proteinase K and trypsin is proof of its proteinaceous nature. In addition, no effect was observed with catalase treatment, evidence that the active agent did not originate from H2O2.

The data obtained in this study indicated that weissellicin L has a completely different protein sequence to other known bacteriocins from Weissella. Additionally, the molecular weight of weissellicin L is smaller than the other four known bacteriocins produced by Weissella sp. (Srionnual et al. 2007; Papagianni and Papamichael 2011; Masuda et al. 2012) (Fig. 5).

As the results showed in the Table 2, bacteriocins from Weissella such as weissellin A, weissellicin Y and weissellicin M were found to have inhibitory abilities against Listeria species except weissellicin 110 (Srionnual et al. 2007; Papagianni and Papamichael 2011; Masuda et al. 2012). Weissellicin L also showed inhibitory ability against Listeria monocytogenes ATCC 19111 (Table 2). The finding here may open future perspective on the use of strain W. hellenica 4-7 to food preservation.

In the previous study of Masuda et al. (2012), it was found that W. hellenica QU 13 could produce two different bacteriocins, termed weissellicin Y and weissellicin M. However, a number of different characteristics were observed among weissellicin L, weissellicin Y and weissellicin M. For example, both weissellicin Y and weissellicin M showed great inhibitory ability against Lactococcus lactis subsp. lactis ATCC 19435T and Bacillus subtilis subsp. subtilis ATCC 6051T. However, no inhibitory activity against these two species was observed with weissellicin L.

When performing BLAST searches of DDBJ databases for partial sequence of weissellicin L, leucocin B-TA33a was the most closely related protein found. It is of great interest that a similar bacteriocin sequence could be observed in two strains belonging to two different genera. Although similar in protein sequence, different inhibitory characteristics were observed. Weissellicin L showed inhibitory activity against Listeria monocytogenes, whereas leucocin B-TA33a did not (Papathanasopoulos et al. 1997). In contrast, leucocin B-TA33a showed inhibitory activity against L. lactis subsp. cremoris, whereas weissellicin L did not (Papathanasopoulos et al. 1997).

The molecular weight of the 17 amino acids was calculated using the Compute pl/Mw tool in the ExPASy Proteomics Server (http://web.expasy.org/compute_pi/), and a result of 1835·14 Da was obtained. However, results from MALDI-TOF-MS indicated that the true molecular weight was approximately 3205·64 Da (Fig. 4). It is therefore considered that more than a dozen of the amino acids remain unknown in this study. To clarify this, an advanced analysis, such as in-gel digestion for mass spectrometric characterization and whole-genome sequencing analysis, will be conducted in the future.

In conclusion, the evidence presented in this report indicates that the W. hellenica isolated from sian-sianzih 4-7 produces a novel bacteriocin, which we have named weissellicin L. Weissellicin L is heat stable and has inhibitory ability against Lmonocytogenes. The application of a specific bacteriocin, or the producer, could result in improved food preservation. Future work in our laboratory will also focus on the possibility of applying this novel bacteriocin as a biopreservative.

Acknowledgements

The authors would like to thank the National Science Council Taiwan for financially supporting this study through a grant to Shwu-fen Pan (Contract No. NSC 101-2313-B-130-001). The authors also thank Mr. Chi-huan Chang for his technical assistance during the amino acid sequence analyses.

Ancillary