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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Leuconostoc sp. J2, isolated from naturally fermented Kimchi, produced a bacteriocin which was named leuconocin J. This bacteriocin exhibited an inhibitory activity against several lactic acid bacteria and some food-borne pathogens. The antimicrobial substance was secreted into the medium during the late log phase. It appears to be proteinaceous since its activity was completely inactivated by a range of proteolytic enzymes, and it was also relatively heat-stable. The bacteriocin was partially purified by ammonium sulphate precipitation, following dialysis. The apparent molecular mass of partially purified bacteriocin, as indicated by activity detection after Tricine-SDS-PAGE, was 2·5–3·5 kDa. Leuconostoc sp. J2 plasmid DNA digested by EcoRI was cloned into pUC118 and transformed into Escherichia coli DH5α. Phenotypic expression of the bacteriocin production was detected in transformants harbouring pULBJ5·5. Finally, Southern blotting with the 2·3 kb insert as a probe against plasmid digests of Leuconostoc sp. J2 revealed that the cloned foreign DNA originated from Leuconostoc sp. J2.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Lactic acid bacteria are widely used as starter cultures for dairy, meat and vegetable fermentations. They contribute to flavour development, as well as preservation of foods, and also produce a variety of compounds with antimicrobial activity, including organic acids, hydrogen peroxide and bacteriocins ( Hoover & Harlander 1993). Bacteriocins are proteinaceous bacterial products which have bactericidal activity against bacteria that are generally closely related species ( Tagg et al. 1976 ). Bacteriocin production among lactic acid bacteria is a widespread phenomenon ( Klaenhammer 1988). Several bacteriocins produced by lactic acid bacteria inhibit not only species closely related to the producer strain but also the growth of pathogenic bacteria, including Listeria monocytogenes, and spoilage bacteria ( Pucci et al. 1988  ; Hechard et al. 1992  ; Cintas et al. 1995 ). Thus, these compounds have attracted increasing interest in improving the quality and safety of various fermented foods ( Gonzalez et al. 1994 ).

Leuconostocs are heterofermentative lactic acid bacteria, found in fermenting vegetables, dairy products and wine ( Garvie 1986). They have been traditionally used with other lactic acid bacteria to preserve food by natural or induced fermentations ( Cogan 1985). Several strains of Leuconostoc spp. produce bacteriocin ( Stiles 1994). Since Orberg & Sandine (1984) reported an antimicrobial substance from Leuconostoc sp. PO184, there have been several reports on leuconostoc bacteriocins, such as leucocin A ( Hastings et al. 1991 ), mesentericin Y105 ( Hechard et al. 1992 ) and mesenterocin 52 ( Mathieu et al. 1993 ). All of the above strains were isolated from meat or dairy products. There have been no studies on bacteriocin production by leuconostocs of plant origin.

A bacteriocin produced by Leuconostoc sp. J2 strain was originally isolated from Kimchi, which is a traditional Korean fermented Chinese cabbage. This study describes the partial characterization of bacteriocin, termed leuconocin J, from Leuconostoc sp. J2. Leuconocin J inhibits the growth of selected strains of lactic acid bacteria and food-borne pathogens. The cloning of the gene coding for leuconocin J is also described.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Bacterial strains, media and plasmids

Bacterial strains and plasmids used in this study are listed in Table 1. As reported earlier, Leuconostoc sp. J2 grew well on the NLS medium ( Choi et al. 1996 ) and showed inhibitory activity against a few Gram-negative and various Gram-positive bacteria. This strain was propagated in lactobacilli MRS broth (Difco) or MRS agar 1·5% (w/v) at 30 °C. For production of bacteriocin, single colonies were inoculated into MRS broth and statically incubated overnight at 30 °C. A 1% (v/v) inoculum of this overnight culture was added to MRS broth. Escherichia coli was grown in LB broth (Difco) or LB agar 1·5% (w/v) at 37 °C. The selective concentration of ampicillin for growing E. coli was 100 μg ml−1. Staphylococcus aureus, used as an indicator strain, was grown in Tryptic Soy Broth (Difco) or Tryptic Soy agar 1·5% (w/v).

Table 1.  Bacterial strains and plasmids used in this study
Bacterial strain or plasmidDescriptionSource or reference
Strains
Leuconostoc sp. J2Lnc+ Imm+ containing native plasmids pLBJ1, pLBJ2 and pLBJ3Isolate
Staphylococcus aureus YSD40Indicator strainOur strain collection
Escherichia coli
DH5αrecA, EndA1, gyrA96, thi-1, hsdR17, supE44, ΔlacU169 (ϕ80, dlacZΔ, M15) deoR FλBRL Life Technologies, (Gaithersburg, MD, USA)
ULBJ5·5E. coli DH5α containing pULBJ5·5 This study
Plasmids
pUC118LacZ′, Ampr, 3·2 kb Vieira & Messing (1987 )
pLBJ1CrypticThis study
pLBJ2CrypticThis study
pLBJ3CrypticThis study
pULBJ5·5pUC118 containing a 2·3 kb EcoRI fragment of Leuconostoc sp. J2This study

Preparation of crude bacteriocin extracts

Leuconostoc sp. J2 was grown to stationary phase statically at 30 °C for 16 h in MRS broth. Cells were removed by centrifugation (5000 g for 10 min at 4 °C). The bacteriocin present in the culture supernatant fluid was concentrated by adding ammonium sulphate (60% saturation) to the crude extract to a concentration of 70% (w/v) and kept overnight at 4 °C with gentle stirring. The precipitate was collected by centrifugation (7000 g for 20 min at 4 °C) and dialysed using a membrane with a 3·5 kDa cut-off (Spectrum, Louna Hills, CA, USA) against 0·01 mol l−1 potassium phosphate buffer (pH 6·6). After dialysis, the solution in the dialysis membrane was sterilized by filtration through a 0·45-μm pore size filter (Millipore). The sterilized samples were stored at −20 °C until further use and were stable for 2 months.

Bacteriocin detection and assay

Bacteriocin was detected by the agar well diffusion method ( Tagg & McGiven 1971). Tryptic soy soft agar 0·7% (w/v), inoculated with 1% (v/v) of an indicator strain Staph. aureus YSD40 overnight culture, was overlaid on tryptic soy agar 1·5% (w/v). Wells were punched in the agar plate and filled with 100 μl of test samples. After incubation overnight at 37 °C, the diameters of the inhibition zones were measured. Leuconocin J activity was assayed by a critical-dilution micro-method using a microtiter plate ( Daba et al. 1993 ). Serial two-fold dilutions of the sample described above were made in 125 μl volumes of tryptic soy broth in a 96-well microtiter plate (Falcon, Fischer Scientific, Pittsburgh, PA, USA). Each well was then inoculated with 50 μl of a 100-fold diluted overnight culture of the test organism Staph. aureus YSD40. Assay microplates were incubated at 37 °C for 18 h. One arbitrary unit (AU) was defined as a 125 μl portion from the highest dilution of the bacteriocin preparation which prevented visible turbidity in a well at 18 h. The activity of the bacteriocin preparation expressed in AU ml−1 was calculated with the formula (1000 · 125−1)×(D−1), where D is the highest dilution that allowed no growth of the test organism at 18 h of incubation.

Sensitivity to heat and proteolytic enzymes

Crude leuconocin J was heated under selected time–temperature conditions, and samples were taken to detect activity. To determine the influence of proteolytic enzymes, the following enzymes (1 mg ml−1) were used (all Sigma) : trypsin, pronase E, α-chymotrypsin and pepsin. Leuconocin J/enzyme mixtures were incubated for 2 h at 37 °C.

Sds-page

The apparent molecular mass of leuconocin J was estimated by the Tricine-SDS-PAGE method ( Schägger & von Jagow 1987). Stacking and separating gels were separated by a spacer gel. The polyacrylamide concentration of the three gels was 4, 10 and 16·5% (w/v), respectively. After electrophoresis at 150 V for 6 h, the gel was removed and cut into two vertical sections. Part of the gel was stained with Coomassie brilliant blue R-250 (Sigma) while the other gel was assayed for antimicrobial activity by direct test as previously described ( Bhunia et al. 1987 ).

Molecular cloning and expression of the leuconocin J gene in E. coli

Plasmid DNA from Leuconostoc sp. J2 was prepared as previously described ( Anderson & McKay 1983). Plasmid DNA from E. coli was isolated by the alkaline lysis method ( Birnboim & Doly 1979). Transformation of E. coli was carried out as described by Sambrook et al. (1989) . Restriction endonucleases and T4 DNA ligase (Boehringer Mannheim) and alkaline phosphatase, calf intestinal (Promega, Madison, WI, USA) were used according to the manufacturers’ recommendations. Restriction fragments were isolated and purified from 0·7% (w/v) agarose gels with a Qiaex II Gel Extract Kit (Qiagen, Valencia, CA, USA). For expression of the bacteriocin production gene in E. coli, plasmid extracts from Leuconostoc sp. J2 were digested with EcoRI and ligated into the EcoRI site of pUC118. The ligated DNA was transformed into E. coli DH5α and positive clones were selected by plating onto LB agar containing X-gal. The bacteriocin production of transformants was tested by using a deferred antagonism assay ( Tagg et al. 1976 ) and the agar well diffusion method. Probes for Southern hybridization were labelled with a digoxygenin (DIG) labelling and detection kit (Boehringer Mannheim). Prehybridization, hybridization and immunological detection were performed as recommended by the supplier. Hybridizations were performed for at least 6 h at 68 °C and post-hybridization washes were performed twice in 2×SSC containing 0·1% SDS at room temperature. Immunological detection was performed with anti-DIG-AP (digoxigenin-alkaline phosphatase) conjugate in the dark.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Leuconostoc sp. J2 was found to produce a bacteriocin, termed leuconocin J. The inhibitory spectrum of leuconocin J was investigated by the agar well diffusion method on several lactic acid bacteria and pathogenic strains ( Table 2). Leuconocin J was inhibitory to growth of a variety of closely related bacteria in the genera Leuconostoc, Lactobacillus, Lactococcus, Pediococcus and other Gram-positive bacteria, including Staph. aureus and L. monocytogenes. More interestingly, leuconocin J was effective against all Gram-negative bacteria tested. When Leuconostoc sp. J2 was grown at different temperatures, maximum production of leuconocin J occurred at 30°C. Leuconocin J production was detected down to 37°C. Leuconocin J production was affected by the pH of the medium. The optimal broth condition for the production of leuconocin J was evaluated under controlled pH conditions. At pH 6·5, the maximum production of leuconocin J was observed at 16 h. A little activity was detected at pH 5·0, 5·5, 6·0 or 7·0. Consequently, cells were grown at an initial pH of 6·5 at 30 °C for 16 h to enhance the yield of bacteriocin ( Fig. 1). Leuconocin J activity was completely eliminated upon treatment with trypsin, pronase E, α-chymotrypsin and pepsin ( Table 3), but was not affected by catalase. After incubation of crude leuconocin J at 62 °C for 30 min, the bacteriocin activity was not reduced, but at 100 °C for 10 min activity was reduced to 20% and at 121 °C for 15 min was inactivated. When stored at 4 °C for 7 d, leuconocin J kept its activity, whereas storage at room temperature was not appropriate, because rapid loss of activity occurred (data not shown). SDS-PAGE was used to estimate the molecular mass of leuconocin J. The gel was stained with Coomassie brilliant blue and other half of the gel, overlaid with the indicator strain, showed that the apparent molecular mass could be estimated in this way to be 2·5–3·5 kDa ( Fig. 2). The EcoRI-digested fragments from Leuconostoc sp. J2 plasmids were ligated to the EcoRI site of pUC118, introduced into E. coli DH5α and screened for their bacteriocin-producing capabilities by using the deferred antagonism and agar well diffusion methods. The transformants harbouring pULBJ5·5 were found to produce an active leuconocin J ( Fig. 3) but controls using non-recombinated E. coli DH5α did not inhibit Staph. aureus (data not shown). Southern blot hybridization analysis showed that this clone had a 2·3 kb EcoRI fragment of plasmid extracts from Leuconostoc sp. J2 ( Fig. 4).

Table 2.  Inhibition spectrum of leuconocin J
Indicator strainNo. of strains inhibited/no. tested *
  1. *Inhibition was tested by the agar well diffusion method ( Tagg & McGiven 1971).

Lactobacillus acidophilus0/1
Lactobacillus cremoris0/1
Leuconostoc paramesenteroides0/1
Lactobacillus casei0/3
Leuconostoc mesenteroides subsp. mesenteroides0/4
Streptococcus thermophilus3/3
Lactobacillus brevis2/2
Lactobacillus plantarum2/2
Leuconostoc mesenteroides subsp. cremoris2/2
Lactobacillus lactis1/1
Lactobacillus fermentum1/1
Lactobacillus helveticus1/1
Leuconostoc lactis1/1
Pediococcus acidilactici1/1
Lactococcus lactis1/1
Listeria monocytogenes1/1
Staphylococcus aureus1/1
Escherichia coli1/1
Serratia marcescens1/1
Yersinia enterocolitica1/1
image

Figure 1.  Growth of Leuconostoc sp. J2 (▪) and production of leuconocin J (▴) in MRS broth at 30 °C

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Table 3.  Sensitivity of leuconocin J to different enzyme and heat treatments
TreatmentDiameter (mm) of zone of inhibition
  1. Indicator strain, Staphylococcus aureus YSD40.

Control20
Pronase E0
α-Chymotrypsin0
Trypsin0
Pepsin0
Catalase20
Lipase20
α-Amylase20
4°C for 7 d20
62°C for 30 min20
85°C for 15 min17
100°C for 10 min16
121°C for 15 min0
image

Figure 2. (a) Coomassie stained half of the Tricine-SDS-PAGE gel, and (b) the other half of the gel, overlaid with Staphylococcus aureus YSD40. Lane 1, Low molecular weight standards (MW-SDS-17, Sigma), 16 950, 14 400, 8160, 6210, 3480 and 2510 Da, lane 2, crude leuconocin J

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image

Figure 3.  Antimicrobial activity of leuconocin J against Staphylococcus aureus YSD40 by the well diffusion method. Wells: a, untreated; b, sample treated with trypsin (1 mg ml−1); c, sample after heating at 62 °C for 30 min; d, sample treated with pronase E (1 mg ml−1), e, Escherichia coli DH5α containing recombinant leuconocin J-producing gene

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image

Figure 4.  (a) Agarose gel electrophoresis and (b) Southern blot hybridization of a 2·3 kb EcoRI fragment of plasmid from Leuconostoc sp. J2 with a leuconocin J-producing specific DNA probe isolated from pULBJ5·5. Lanes: M, λDNA/HindIII marker (23·1, 9·4, 6·6, 4·4, 2·3, 2·0 and 0·6 kb) and negative control; A, Leuconostoc sp. J2 plasmid DNA digested by EcoRI

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This study dealt with the isolation and partial characterization of leuconocin J and the cloning and expression of the bacteriocin-producing gene in E. coli. Leuconostoc sp. J2, isolated from Kimchi, produced a proteinaceous substance with antimicrobial activity, which was secreted from cells principally during the late exponential phase of growth. The activity reduced gradually during the stationary phase. A similar observation was reported for other bacteriocins, such as pediocin AcH ( Biswas et al. 1991 ), helveticin J ( Joerger & Klaenhammer 1986) and lactocin S ( Mortvedt & Nes 1990), suggesting that the antimicrobial substance was a secondary metabolite.

Crude leuconocin J was prepared by ammonium sulphate precipitation. It inhibited the growth of a large number of other lactic acid bacteria, some food-borne pathogens and, especially, Gram-negative bacteria. Generally, under normal conditions, Gram-negative bacteria are not sensitive to bacteriocins produced by Gram-positive bacteria ( Jack et al. 1995 ). Although the cytoplasmic membrane should be susceptible, the outer membrane protects the cell from bacteriocins. The outer membrane of Gram-negative bacteria acts as a permeability barrier for the cell. It prevents molecules such as antibiotics (or bacteriocins), detergents and dyes from reaching the cytoplasmic membrane ( Stevens et al. 1991 ). In contrast, leuconocin S ( Lewus et al. 1992 ), produced by Leuconostoc paramesenteroides OX, was active against Yersinia enterocolitica and lactocin 705 ( Vignolo et al. 1993 ), produced by Lactobacillus casei CRL 705, was also active against a wide range of Gram-negative bacteria. However, the mechanism of action has not yet been demonstrated.

Leuconocin J was proteinaceous because its activity was inactivated by treatment with proteolytic enzymes but was not affected by heat treatment (62 °C for 30 min) and catalase. These results suggested that the inhibitory effect was not attributable either to pH, organic acid or hydrogen peroxide produced by the culture.

The molecular mass of leuconocin J was 2·5–3·5 kDa by a direct activity assay as described by Bhunia et al. (1987) after Tricine-SDS-PAGE ( Schägger & von Jagow 1987). Leuconocin J was retained in a 3·5 kDa cut-off dialysis membrane. Therefore, it seems to aggregate with other substances or to aggregate into its polymer forms in non-denaturing conditions. Low molecular weight bacteriocins were reported for several other bacteriocins such as 5·8 kDa lactococcin A ( Holo et al. 1991 ), 2·7 kDa pediocin AcH ( Bhunia et al. 1988 ), 3·9 kDa leucocin A ( Hastings et al. 1991 ) and 3·5 kDa divergicin 750 ( Holck et al. 1996 ).

Curing studies with acriflavine and acridine orange did not produce a bacteriocin non-producing phenotype. Therefore, shot-gun cloning was performed and yielded one bacteriocin-producing clone. In recent years, a number of bacteriocins have been cloned and sequenced but few reports deal with genetic characterization in leuconostocs ( Hastings et al. 1991  ; Fremaux et al. 1995  ; van Belkum & Stiles 1995). We are currently investigating purification and expression of the bacteriocin in other lactic acid bacteria and also characterizing leuconocin J at the molecular level.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This investigation was supported in part by a research grant from the Korea Research Foundation 1995.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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