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Brevicin 174Aγ-like novel bacteriocin produced by Lactobacillus brevis JP0063 isolated from kimchi§
Korean J. Microbiol. 2023;59(4):307-318
Published online December 31, 2023
© 2023 The Microbiological Society of Korea.

Jae-Yong Park*

Department of Food Science and Nutrition, Daegu Catholic University, Gyengsan 38430, Republic of Korea
Correspondence to: *E-mail: jaepark@cu.ac.kr; Tel.: +82-53-850-3521; Fax: +82-53-359-6580
§Supplemental material for this article may be found at http://www.kjom.org/main.html
Received November 29, 2023; Revised December 14, 2023; Accepted December 15, 2023.
Abstract
To isolate bacteriocin-producing lactic acid bacteria (LAB) that inhibit Lactobacillus plantarum, which can induce kimchi spoilage, a strain of L. plantarum was first isolated from kimchi and used as an indicator. The isolate was identified as L. plantarum through 16S rRNA sequencing and recA multiplex PCR analysis and was named L. plantarum JP0059. An LAB strain that inhibits JP0059 was isolated from kimchi, identified as Lactobacillus brevis through 16S rRNA sequencing, and named L. brevis JP0063. Lactobacillus brevis JP0063 showed maximum production of bacteriocin in the exponential and stationary phases. This bacteriocin primarily inhibited homolactic fermentable Lactobacillus, but not Leuconostc. The bacteriocins were partially purified from the culture supernatant using a cation-exchange column. SDS-PAGE confirmed that the partially purified bacteriocin had a molecular weight of approximately 5 kDa and was relatively stable under pH and heat treatment. However, the bacteriocin activity disappeared after proteinase K treatment. Bacteriocins produced by JP0063 inhibited JP0059 through bactericidal action. The N-terminal amino acid sequence of the bacteriocin was analyzed as KKKKKVACTWGNAAAA, which was highly homologous to that of brevicin 925A-γ produced by L. brevis 925A. Primers were designed based on the brevicin 925A-γ neighboring genes, and PCR amplification and sequencing was performed; two bacteriocin genes were found. The amino acid sequences deduced from the bacteriocin genes were compared with those of brevicin 925A. In the breB gene, a single amino acid in the signal peptide varied, whereas in breC gene, three amino acids in the bacteriocin structural gene varied.
Keywords : Lactobacillus brevis, brevicin, kimchi, novel bacteriocin
Body

Kimchi, a traditional Korean fermented food, is naturally fermented by various lactic acid bacteria (LAB), including Leuconostoc, Weissella, and Lactobacillus (Jung et al., 2011). According to previous studies on the kimchi ecosystem, the genus Leuconostoc of LAB predominate in the early and middle stages of kimchi fermentation, producing a variety of metabolites, including CO2, acetic acid, sugar alcohols, and lactic acid, by heterolactic fermentation (Han et al., 1990). During these fermentation stages, the metabolism of the genus Leuconostoc plays a crucial role in developing the unique flavor of kimchi. As fermentation progresses, it is exposed to a lower pH environment unsuitable for the growth of the genus Leuconostoc, which is relatively less acid-tolerant than homolactic LAB. In the late stage of kimchi fermentation, the dominant taxa change to homolactic fermentable Lactobacillus, such as Lactobacillus plantarum, and these species produce excessive acids, resulting in an off-flavor and acidification of kimchi. During this process, acid-tolerant yeasts and molds proliferate, decreasing the kimchi quality (Han et al., 1990). This hypothesis for a shift in the dominant microflora during kimchi fermentation is valid when molecular biology techniques are applied to study the transition of microorganisms during kimchi fermentation (Jung et al., 2011; Park et al., 2012). This means inhibiting homolactic fermentable Lactobacillus growth may improve the kimchi shelf life.

Bacteriocins are antimicrobial peptides produced naturally by bacteria and can be digested by human digestive proteases. This feature ensures safety for the human body by preventing the residues of antimicrobial substances. Bacteriocins inhibit bacteria species that occupy the same ecological position or are closely related to producer strains (Deegan et al., 2006). Bacteriocins are expected to be an adequate replacement for chemical preservatives because, unlike antibiotics, they can attack specific target microorganisms while maintaining a LAB that is beneficial to human health and is generally recognized as safe (GRAS) (Deegan et al., 2006; Settanni and Corsetti, 2008; Yang et al., 2014). The representative bacteriocin nisin, produced by Lactococcus lactis, has been officially approved as a food preservative in several countries and is extensively used in the food industry (Deegan et al., 2006; Settanni and Corsetti, 2008). Therefore, bacteriocins with inhibitory activity against pathogenic bacteria can be useful additives to improve food safety.

Bacteriocins have been investigated as potential acidification control agents. When bacteriocin-producing Enterococcus faecium DU0267 isolated from kimchi was used as a starter, the Lactobacillus count was substantially lower than that of the control group and gas production was reduced to 60% of that of the control during kimchi fermentation (Ha and Cha, 1994). Enterococcus sp. K25, which produces bacteriocins that inhibit L. plantarum, has a synergistic effect on extending the shelf life of kimchi when fumaric acid is added as a starter (Moon et al., 2004). In addition, when Leuconostoc citreum GJ7, which produces bacteriocin, was used as a fermentation starter for kimchi, incubated at 7°C for 12 to 15 days and stored at -1°C, no yeast appeared after 125 days, effectively preventing the overripening of kimchi. However, it appeared after 50 days in kimchi without the starter (Chang and Chang, 2010). Kimchi fermented with Leu. citreum GR1 strain, which had enhanced bacteriocin production ability in the presence of L. plantarum cell faction, had higher sensory quality and extended shelf life (Moon et al., 2018). The above reports show that kimchi prepared with LAB, which produces bacteriocins that inhibit the genus Lactobacillus, such as L. plantarum, prolonged the shelf life as a starter culture.

In this study, bacteriocin-producing LAB was isolated from kimchi using L. plantarum isolated from kimchi as an indicator instead of reference strains. This bacteriocin is expected to prolong the shelf life of kimchi by inhibiting the L. plantarum strain in kimchi. Additionally, we characterized the bacteriocin-producing LAB, partially purified bacteriocin, and identified it as a novel bacteriocin with differences in the amino acid sequence from previously reported bacteriocins.

Materials and Methods

Isolation and identification of an indicator strain

Since the acidity of kimchi at the optimum ripening stage is known to be 0.4–0.9% (Park et al., 2017), acidified kimchi (acidity < 0.39%) samples were serially diluted, spread on an MRS (Difco Laboratories) agar, and cultivated at 30°C for 24 h. The colonies were inoculated into MRS broth, and chromosomal DNA was extracted from each colony. The extracted DNA was used to amplify the 16S rRNA gene using the primer 27f (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1525r (5’-AAGGAGGTGWTCCARCC-3’). PCR (T100TM Thermal Cycler, Bio-Rad) conditions were as follows: 30 cycles of denaturation at 94°C for 30 sec, annealing at 54°C for 30 sec, and extension at 72°C for 60 sec. The amplified 16S rRNA gene was cloned into a T-easy vector (Promega), and the DNA sequence was determined using the dideoxy chain termination method at COSMO Genetech. Homology of the 16S rRNA sequence was analyzed using NCBI BLAST, and a strain identified as L. plantarum was selected. For a more precise taxonomic identification, a recA multiplex PCR assay was performed to distinguish L. plantarum, Lactobacillus paraplantarum, and Lactobacillus pentosus, which are closely related genetically and exhibit highly similar phenotypes (Torriani et al., 2001). PCR conditions were 30 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, and extension at 72°C for 30 sec using four primers (paraF, 5’-GTCACAGGCATTACGAA AAC-3’; pentF, 5’-CAGTGGCGCGGTTGATATC-3’; planF, 5’-CCGTTTATGCGGAACACCTA-3’; pREV, 5’-TCGGGA TTACCAAACATCAC-3’) (Torriani et al., 2001).

Isolation and identification of the bacteriocin-producing strain

Kimchi samples were serially diluted, spread on an MRS agar, cultivated at 30°C for 24 h, and overlaid with 0.75% MRS soft agar containing the indicator L. plantarum strain. After overnight incubation at 30°C, bacteriocin-producing strains were selected by a clear zone of inhibition around the colony. The antimicrobial activity was tested using the spot-on-the-lawn method (Moraes et al., 2010). The produced substance was confirmed to be a bacteriocin after treatment with proteinase K. The 16S rRNA gene sequence analysis identified the bacteriocin-producing strain.

Growth curve and bacteriocin activity

The bacteriocin-producing strain was cultivated in MRS broth at 30°C. The absorbance of the culture was measured for 24 h at 600 nm by using a UV spectrophotometer (JASCO V-530). Simultaneously, the culture supernatant was prepared by centrifugation (30,000 × g, 4°C, 10 min) and filtration (Sartorius 0.20 μm syringe filter). Bacteriocin activity was detected in the culture supernatant using the well-diffusion method on an MRS plate (Magaldi et al., 2004). The bacteriocin activity was determined by comparing the diameters of the inhibition zones.

Inhibitory spectrum

The inhibitory spectrum of the bacteriocins was determined using various LAB and pathogenic bacteria as indicators using the spot-on-the-lawn method. First, an overnight culture of the bacteriocin-producing strain was spotted onto MRS plates. It was then overlaid with 0.75% MRS soft agar containing LAB as an indicator strain. For the pathogenic bacteria, the culture medium consisted of 0.75% BHI soft agar (Listeria), 0.75% LB soft agar (Escherichia), and 0.75% nutrient soft agar (Salmonella, Bacillus, and Klebsiella). After overnight incubation at 30°C or 37°C, the size of the clear zone of inhibition was determined.

Partial purification of the bacteriocin

The culture supernatant was obtained by filtering through a 0.45 μm-pore size membrane filter (Advantec MFS) from the overnight culture of the bacteriocin-producing strain. The purification devices used were a 5 ml HiTrap SP Sepharose Fast Flow Cation-exchange Column (GE Healthcare) and the ÄKTAprime Plus System (GE Healthcare) (Uteng et al., 2002). The resulting supernatant (500 ml) was loaded onto a purification column. The starting buffer (binding buffer) contained 50 mM sodium phosphate (pH 7.0). The samples were eluted using a linear NaCl gradient (0-1 N) by mixing 1N NaCl with the starting buffer (Kim et al., 2004b). The flow rate was adjusted to 5 ml/min and 5 ml of each fraction was collected. Each eluted fraction was examined for bacteriocin activity using the spot-on-the-lawn method. Fractions with high activity were collected, desalinated, and concentrated using an Amicon Ultra-4 centrifugal filter (Millipore). Partially purified bacteriocin activity, expressed as activity units (AUs) per ml was defined as the reciprocal of the highest two-fold dilution still showing inhibitory action towards the indicator organism.

SDS-PAGE and activity staining of the partially purified bacteriocin

To confirm the purification, the partially purified bacteriocin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a Tris-Tricine buffer system (Schägger and von Jagow, 1987). The electrophoresis gel contained 16.5% T and 3% C acrylamide-bisacrylamide, and the electrophoresis conditions were 30 mA for approximately 2 h. After gel electrophoresis, half of the gel was used to determine the molecular weight of the bacteriocin by silver staining using a Silver Stain Plus Kit (Bio-Rad). The other half was washed with deionized water for 4 h and overlaid with 0.75% MRS soft agar containing an indicator strain (L. plantarum JP0059) on an MRS plate (Bhunia et al., 1987). After overnight incubation at 30°C, the zone of inhibition was examined and compared with the stained bacteriocin band.

Analysis of stability of the partially purified bacteriocin

The stability of the bacteriocin was examined against pH, heat, and enzyme treatments. First, the same amount of diluted partially purified bacteriocin was mixed with a buffer solution (pH 2.5 to 9.5) (Yang et al., 2002). To examine heat stability, the partially purified bacteriocin was incubated at 60°C, 80°C, and 100°C for up to 60 min, respectively. It was also subjected to autoclaving (121°C, 15 min) The enzymes used were trypsin (bovine pancreas Type Ⅰ, Sigma), lipase (Candida rugose Type VII, Sigma), protease (Streptomyces griseus Type X IV, Sigma), lysozyme (egg white, Amresco), pepsin (porcine gastric mucosa, Sigma), proteinase K (TaKaRa), and α-amylase (Aspergillus oryzae, Sigma). Enzymes were prepared as 20 mg/ml stock solutions and added to the partially purified bacteriocins at a final concentration of 4 mg/ml enzyme (Yang et al., 2002). The enzyme mixtures were incubated at 37°C for 12 h. After treatment with heat, pH buffer, and enzymes, the residual bacteriocin activity was measured using the spot-on-the-lawn method.

Mode of action of the bacteriocin

The indicator strain, L. plantarum JP0063, was prepared by incubation in MRS broth for 8 h. The cells were washed and suspended in 3 ml 50 mM Tris-HCl buffer (pH 8.3). After adding the diluted partially purified bacteriocin (1,600 AU/ml), the cells were incubated at 30°C, and the number of viable cells was measured at 3 h intervals (Moon et al., 2000; Kim et al., 2004b). Bacteriocins were not added to the control group. The cells in the other group were treated with partially purified bacteriocin (1,600 AU/ml) and proteinase K (4 mg/ml). The number of viable cells was determined using the poured-plate method.

N-Terminal amino acid sequence analysis

N-Terminal sequence analysis detected the amino acid sequence of the bacteriocin. First, SDS-PAGE was performed as described above. After gel electrophoresis, the partially purified bacteriocins were transferred to a PVDF membrane (Bio-Rad) and the PVDF membrane was stained with Coomassie Brilliant Blue R-250 (Amresco). The stained bacteriocin bands were cut and dried. N-terminal peptide sequence analysis of the bacteriocin was performed using Edman degradation (Edman, 1949; Reim and Speicher, 1997) at SIS Corp.

DNA amplification, cloning, and sequencing

Primers were designed for bacteriocin gene amplification based on the plasmid sequence of L. brevis 174A (Noda et al., 2015) (NCBI Accession #NC_012551). The bacteriocin gene was amplified with L. brevis JP0063 plasmids using the primers JYP99 (5’-CCCGCGACCAAAATACTAAG-3’) and JYP100 (5’-GGATCGTTTGTTGCCGCTT-3’). PCR conditions were 30 cycles of denaturation at 94°C for 30 sec, annealing at 54°C for 30 sec, and extension at 72°C for 60 sec. The PCR amplification product was cloned into the T-easy vector (Promega) and sequenced.

Nucleotide sequence accession number

The sequence reported here was deposited in the GenBank database under accession number MF9838558.

Results and Discussion

Isolation and identification of the indicator strain and bacteriocin-producing LAB

To isolate LAB strains that produce bacteriocins that inhibit the growth of L. plantarum in kimchi, L. plantarum ATCC 8014 and ATCC 10241 strains were used as indicators. These two strains were isolated from corn silage and sauerkraut, respectively. It was assumed that the LAB present in kimchi would not frequently encounter each other because of the differences in their growing environments. Therefore, it was thought that the bacteriocin-producing lactic acid bacteria that inhibit these two strains might be less likely to be present in kimchi. Consequently, it was decided that using L. plantarum strains isolated from kimchi as indicator bacteria would be more effective in isolating bacteriocin-producing lactic acid bacteria that could improve the shelf life of kimchi. The shelf life of standard kimchi is approximately one month, and the quality retention period is 60 to 120 days, depending on the storage temperature. The isolated strains were identified by 16S rRNA sequence analysis and recA multiplex PCR assay. The 16S rRNA sequence of one strain was highly similar (99% match) to that of L. plantarum and L. pentosus strains. recA has been used to identify three species (L. plantarum, L. paraplantarum, and L. pentosus) in the L. plantarum group (Torriani et al., 2001). RecA is a small protein that facilitates complex biochemical processes in microorganisms (Eisen, 1995). Accordingly, the recA gene is shared among microorganisms, and its gene product is used as a phylogenetic marker for related strains (Torriani et al., 2001). A recA multiplex PCR assay can be used to obtain amplification products for specific strains by using a species-specific primer mix (L. plantarum KCTC3104, planF, pREV; L. paraplantarum C7, paraF, pREV; L. pentosus ATCC8041, pentF, pREV) (Torriani et al., 2001). The amplification product differed in size among the three strains (Supplementary data Fig. S1). The recA gene of the indicator strain (JP0059) was approximately 300 bp in length and was the same size as the recA of L. plantarum KCTC3104. Therefore, the indicator strain was identified as L. plantarum and named L. plantarum JP0059. This strain was used as an indicator of bacteriocin-producing strains.

A strain that inhibits L. plantarum, JP0059, was isolated from kimchi. The 16S rRNA sequence of this strain is highly similar (sequence similarity of 99%) to the 16S rRNA gene sequence of L. brevis. Therefore, this strain was identified as L. brevis and named L. brevis JP0063. The cell and culture supernatants of L. brevis JP0063 inhibited L. plantarum JP0059 growth. The antibacterial activity of the culture supernatant disappeared after the addition of proteinase K (Supplementary data Fig. S2), indicating that it is proteinaceous. Therefore, it is regarded as a bacteriocin. The bacteriocin produced by L. brevis JP0063 was renamed as brevicin JP0063.

Bacteriocin activity in the growth of L. brevis JP0063

Lactobacillus brevis JP0063 entered the exponential growth phase after 6 h of cultivation at 30°C in MRS media. The strain reached stationary phase after 14 h of cultivation. Lactobacillus brevis JP0063 did not produce brevicin JP0063 in the lag phase and showed the highest bacteriocin production in the exponential and early stationary phases (Fig. 1). Brevicin JP0063 production decreases in the middle of the stationary phase. Most bacteriocins are produced during the exponential growth phase, with peak production occurring during the stationary phase (Kwark et al., 1999; Ko and Ahn, 2000; Yang et al., 2002; Kim et al., 2003; Ahn et al., 2012). After the middle of the stationary phase, bacteriocin activity decreases because of increased proteolytic enzymes in dead cells (Yang et al., 2002; Ahn et al., 2012). The induction of the production of bacteriocins by co-culture with indicator strains has been demonstrated in several examples of LAB (Man et al., 2012; Ge et al., 2014; Piazentin et al., 2022). Despite these findings, the fundamental mechanisms underlying the production or enhancement of bacteriocins by co-culture remain unknown (Chanos and Mygind, 2016).

Fig. 1. Growth and bacteriocin activity of L. brevis JP0063 in MRS (Man, Rogosa, and Sharpe) media at 30°C.
Bacteriocin activity was detected from the culture supernatant using the well-diffusion method on MRS plates.

The inhibitory spectrum of brevicin JP0063

The inhibitory spectrum of brevicin JP0063 was investigated using various LAB and pathogenic bacteria as indicators (Table 1). The inhibition spectrum of brevicin JP0063 was narrow. It mainly inhibited only homolactic fermenting LAB such as Lactococcus cremoris, L. plantarum, Lactobacillus acidophilus, and Lactobacillus casei, but not the genus Leuconostoc, suggesting the applicability of brevicin JP0063 as an additive to improve the shelf life of kimchi. However, since kimchi has a complex ecosystem of microorganisms, it is difficult to conclude that inhibiting the homolactic LAB would prolong the shelf life of kimchi. To overcome these limitations, it is necessary to investigate the preservation period further when adding brevicin JP0063 at the beginning of kimchi fermentation or using L. brevis JP0063 as a kimchi starter culture.

Inhibitory spectrum of the bacteriocin produced by L. brevis JP0063
Indicator Inhibition zonea
Lactococcus cremoris MG1363 +
Lactococcus lactis subsp. lactis ATCC4962 -
Leuconostoc mesenteroides SY1 -
Leuconostoc citreum ATCC49370 -
Leuconostoc lactics ATCC19256 -
Leuconostoc carnosum KCTC3525 -
Leuconostoc mesenteroides ATCC8293 -
Leuconostoc citreum JP0057 -
Leuconostoc citreum JP0058 -
Lactobacillus acidophilus KFRI217 +
Lactobacillus acidophilus ATCC4962 +
Lactobacillus plantarum KCTC3104 +
Lactobacillus plantarum KCTC1048 -
Lactobacillus casei YIT9018 +
Lactobacillus casei ATCC4646 +
Lactobacillus brevis 2.14 -
Lactobacillus paraplantarum C7 -
Lactobacillus plantarum JP0059 +
Lactobacillus plantarum JP0060 +
Listeria monocytogenes ATCC19111 -
Escherichia coli O157:H7 -
Salmonella typhimurium TA98 -
Salmonella typhimurium TA100 -
Bacillus cereus ATCC 14579 -
Klebsiella pneumonia subsp. pneumoniae KCTC1560 -

a Inhibition zone of the cell culture of L. brevis JP0063 against indicators.

-, negative; +, < 2 mm; ++, ≥ 2 mm.



It did not inhibit pathogenic bacteria, such as Listeria monocytogenes ATCC 19111, Escherichia coli O157:H7, Salmonella typhimurium TA98, S. typhimurium TA100, Bacillus cereus ATCC 14579, and Klebsiella pneumonia subsp. pneumoniae KCTC 1560 (Table 1). Most bacteriocins produced by LAB isolated from kimchi exhibit a narrow spectrum. In the case of bacteriocins that inhibit Listeria, the inhibition spectrum of these bacteriocins is narrow (Kim et al., 2003); with some bacteriocins only inhibiting Listeria (Kim et al., 2004b). In the case of LAB inhibition, these bacteriocins have narrow spectra and mostly inhibit Lactobacillus species (Jo et al., 1997).

Partial purification of brevicin JP0063

Brevicin JP0063 was purified from the culture supernatant using cation-exchange chromatography because most bacteriocins produced by LAB are class II, cationic, and hydrophobic in nature (Nes and Holo, 2000). After cation-exchange chromatography, six fractions (each 5 ml) with high bacteriocin activity were pooled. After desalting and concentrating, the partially purified bacteriocin showed a 128-fold increase in specific activity, with 76.8% yield (Supplementary data Table S1).

SDS-PAGE was performed and silver-stained, and brevicin JP0063 was successfully purified, as only a single band of approximately 5 kDa was detected without bands from other proteins, even after partial purification using a cationic column. The inhibition zones for the indicator strains detected under the same electrophoresis conditions appeared at the same positions as the electrophoretic bands (Fig. 2). Previously reported bacteriocins produced from kimchi have a molecular weight of approximately 3–5 kDa (Kim et al., 2004a; Lee et al., 2007; Yang et al., 2014), which is similar to that of brevicin JP0063 detected in this study.

Fig. 2. Mode of inhibitory action of the bacteriocin produced by L. brevis JP0063.
Black circle, no addition of bacteriocin to the suspension of the indicator L. plantarum JP0059; black square, addition of the partially purified bacteriocin (1,600 AU/ml) to the suspension of the indicator cells; open square, simultaneous addition of proteinase K and the partially purified bacteriocin to the suspension of the indicator cells.

Stability of the partially purified brevicin JP0063

The stability of partially purified brevicin JP0063 was investigated under various pH, heat, and enzyme treatments. The bacteriocin activity was not affected by pH variation from 2.5 to 9.5 (Table 2). Our results are consistent with those obtained for other bacteriocins produced by LAB in kimchi, which are stable over a wide pH range (2.0–10.0) (Ko and Ahn, 2000; Yang et al., 2002; Kim et al., 2004a; Ahn et al., 2012). Other bacteriocins have been reported to be stable in acidic or neutral pH ranges, but unstable under alkaline conditions (Jo et al., 1997; Kwark et al., 1999; Lee et al., 2007).

Stability of the partially purified bacteriocin against various treatments
Treatment Residual activity (AU/ml)
Control 16,000
pHa pH 2.5 16,000
pH 4.5 16,000
pH 6.0 16,000
pH 7.0 16,000
pH 8.0 16,000
pH 9.5 16,000
Heat 60°C, 10 min 16,000
60°C, 30 min 16,000
60°C, 60 min 16,000
80°C, 10 min 16,000
80°C, 30 min 16,000
80°C, 60 min 16,000
100°C, 10 min 2,000
100°C, 30 min 2,000
100°C, 60 min -
121°C, 15 min -
Enzymeb Trypsin -
Lipase 16,000
Protease -
Lysozyme 16,000
Pepsin -
Proteinase K -
α-Amylase 4,000

a pH 2.5–4.5, Citric Acid – Sodium Citrate Buffer Solution; pH 6.0–8.0, Na2HPO4–NaH2PO4 Buffer Solution; pH 9.5, Sodium Carbonate–Sodium Bicarbonate Buffer Solution.

b Trypsin (from bovine pancreas Type Ⅰ, Sigma), Lipase (from Candida rugosa Type VII, Sigma), Protease (from Streptomyces griceus Type XIV, Sigma), Lysozyme (from Egg white, Amresco), Pepsin (from porcine gastric mucosa, Sigma), Proteinase K (TaKaRa), α-Amylase (from Aspergillus oryzae, Sigma)



Thermostability of brevicin JP0063 was examined with respect to temperature and time. The activity of brevicin JP0063 was stable after 60 min of treatment at 60°C and 80°C, and 12.5% of the initial activity remained after 10 min of treatment at 100°C. The activity was lost after 60 min of treatment at 100°C or 15 min at 121°C, which are autoclave conditions (Table 2). Previous reports have shown that some bacteriocins retain activity even at high temperatures, such as autoclave conditions (121°C, 15 min) (Kwark et al., 1999; Yang et al., 2002; Kim et al., 2003; Ahn et al., 2012). For some bacteriocins, activity is not fully retained during heat treatment; for example, the activity of lacticin YH-10 was reduced to 50% after treatment at 100°C for 60 min (Park et al., 2004), and paraplantaricin C7 retained 50% of its activity under autoclave conditions (Lee et al., 2007). Bacteriocins produced by L. sakei P3-1 retained 50% and 12.5% activity after treatment at 100°C and autoclave conditions, respectively (Kim et al., 2004b). The results show that brevicin JP0063 is stable at temperatures below 80°C but unstable at temperatures above 80°C, suggesting that brevicin JP0063 is thermostable but not as stable as other previously reported bacteriocins.

Bacteriocin activity was lost after treatment with proteolytic enzymes such as trypsin, protease, pepsin, and proteinase K, whereas total activity was maintained after treatment with lysozyme and lipase (Table 2). Unexpectedly, bacteriocin activity was reduced by 75% after α-amylase treatment, suggesting that brevicin JP0063 is a natural glycoprotein. Although class IV bacteriocins with carbohydrate moieties are controversial, there have been consistent reports of amylase-sensitive bacteriocins (Lewus et al., 1992; Keppler et al., 1994; Seo et al., 2014). However, recent bacteriocin classifications have excluded class IV bacteriocins (Kumariya et al., 2019; Zimina et al., 2020), and further studies are required to determine whether brevicin JP0063 is a glycoprotein.

Mode of action of the brevicin JP0063

Viable cells were counted after addition of partially purified brevicin JP0063 to a suspension of the indicator strain (Fig. 3). The number of viable cells in the indicator strain decreased by 99.9% during the first 3 h and gradually decreased thereafter. When a mixture of brevicin JP0063 and protease K was added to the indicator strain suspension, the number of viable cells remained unaffected. These results show that the proteolytic enzyme eliminated the inhibitory effect of brevicin JP0063. As a result, the inhibitory mode of action of brevicin JP0063 was a bactericidal effect.

Fig. 3. SDS-PAGE and activity detection of the partially purified bacteriocin using silver-stained Tricine-SDS PAGE.
Lanes: M, Size marker (Precision Plus Protein Dual Xtra Standards, Criterion 10–20% Tris-Tricine; BIO-RAD); 1, size band of the partially purified bacteriocin; 2, gel overlaid with MRS agar containing L. plantarum JP0059.

Characterization of the brevicin JP0063 gene

The N-terminal amino acid sequence of brevicin JP0063, identified by Edman degradation, was N-KKKKVACTWGN AAAA. The result of a local alignment search using NCBI protein BLAST showed identity scores of 94% with the N-terminal regions of brevicin 925A (Wada et al., 2009), plantricin 1.25β (Ehrmann et al., 2000) and brevicin 174Aγ (Noda et al., 2015). These three bacteriocins share identical amino acid sequences. Brevicin 925A and 174A-γ and related genes were located in a plasmid, and plantricin 1.25β was chromosomal. Therefore, we expected that the brevicin JP0063 gene would most likely be present in the plasmid of L. brevis JP0063. The agarose gel electrophoresis profile suggested that L. brevis JP0063 contains at least two plasmids (Fig. 4A). To amplify the brevicin JP0063 structural gene, primers were designed based on the brevicin 174A biosynthetic gene cluster (GenBank accession number: LC062087.1) and PCR was performed using the extracted plasmid DNA as a template. The nucleotide sequence of the 1 kb DNA fragment was identical to the breB, breC, and breD sequences of L. brevis 174A (Fig. 4B). The deduced amino acid sequence of breC matched the N-terminal amino acid sequence (KKKKVACTWGNAAAA) of partially purified brevicin JP0063 (Fig. 4C), indicating that breC is the structural gene of brevicin JP0063. The deduced amino acid sequence of the breB gene of L. brevis JP0063 showed one substitution in the leader sequence compared with the breB gene encoding brevicin 174Aβ. The deduced amino acid sequence of the breC gene of L. brevis JP0063 showed one deletion and two differences compared with the amino acid sequence deduced from the breC gene encoding brevicin 174Aγ (Fig. 4C). These results suggest that the bacteriocin produced by L. brevis JP0063 is similar to brevicin174Aβ and 174Aγ but is a novel bacteriocin.

Fig. 4. Plasmid profile of L. brevis JP0063 and the structural gene cluster of brevicin JP0063.
(A) 1% Agarose gel electrophoresis of plasmids of L. brevis. Lanes: M, GeneRuler 1 kb DNA ladder (Thermo Fisher); 1, plasmid profile. (B) Gene cluster of breC and its neighboring genes. (C) Amino acid sequence comparison of brevicin JP0063-β with 174A-β, JP0063-γ, and 174-γ. Differences in amino acid sequence are colored in gray. The predicted double glycine at the end of the signal peptide is represented by a box.

The bacteriocins encoding breB and breC from L. brevis JP0063 were renamed brevicin JP0063β and brevicin JP0063γ, respectively. The putative promoter sequence was located upstream of breB, and the putative terminator was located downstream of breC. These findings indicate that breB and breC form operons. The antibacterial activity of brevicin174Aβ and 174Aγ was increased up to 100-fold when treated in a 1:1 mixture compared to each other (Noda et al., 2015). In addition, brecicin174Aβ and 174Aγ also have GXXXG in the mature sequence, a typical motif found in class IIb bacteriocins (Nissen-Meyer et al., 2009). This motif may mediate helix-helix interactions between bacteriocins (Class IIb) by interhelical van der Waals interactions and hydrogen bonding in enemy cell membranes (Senes et al., 2004). There were no stability test data for enzymatic or heat treatments for bacteriocins with very similar sequences to brevicin JP0063β and JP0063γ. However, the reported inhibition spectra were similar to those of brevicin JP0063. Therefore, brevicin JP0063β and JP0063γ may also be class 2b bacteriocins that must form a complex to be active.

Poly-lysine residues were located in the N-terminal region of mature brevicin JP0063β and JP0063γ; these regions may attach themselves to enemy cells' negatively charged membrane surface (Nissen-Meyer et al., 2009). The putative transcriptional regulator (encoding breD) contained a helix-turn-helix motif (COG1476) and showed two amino acid substitutions compared to the sequence encoded by breD in L. brevis 174A. The brevicin gene cluster of L. brevis 925A and 174A contained two transcriptional regulator-encoding genes (breD and breG) with helix-turn-helix motifs. However, it is unclear which genes are regulated by these two factors.

Despite the similarity in amino acid sequence and similarity in gene structure, there is still a lack of clear evidence suggesting that the mechanism of action of brevicin JP0063β and JP0063γ will function similarly to brevicin 174Aβ and 174Aγ. Further research on the mechanism of action of the genes in this bacteriocin gene cluster, whether they function as glycoproteins, and their applicability to kimchi strains is required.

적 요

김치 산패를 유발할 수 있는 Lactobacillus plantarum을 억제하는 박테리오신 생성 유산균을 분리하기 위해 먼저 김치에서 L. plantarum 균주를 분리하여 지표균으로 사용하였다. 분리된 균주는 16S rRNA 염기서열 분석과 recA multiplex-PCR 분석을 통해 L. plantarum으로 확인되었으며, L. plantarum JP0059로 명명하였다. L. plantarum JP0059를 억제하는 유산균주는 김치에서 분리되어 16S rRNA 염기서열 분석을 통해 Lactobacillus brevis로 확인되었으며, L. brevis JP0063으로 명명하였다. L. brevis JP0063은 지수 및 정지 단계에서 박테리오신을 최대로 생산하는 것으로 나타났다. 이 박테리오신은 주로 유산균을 억제했지만 Leuconostoc은 억제하지 않았다. 양이온 교환 컬럼을 사용하여 배양 상층액에서 박테리오신을 부분정제하였다. 부분정제된 박테리오신의 분자량은 약 5 kDa이며 pH와 열처리에서도 비교적 안정하다는 것을 SDS-PAGE를 통해 확인되었으며, 박테리오신 활성은 프로테아제 K 처리 후 사라졌다. L. brevis JP0063이 생성한 박테리오신은 살균 작용을 통해 L. plantarum JP0059를 억제했다. 박테리오신의 N-말단 아미노산 서열을 분석한 결과, L. brevis 174A 가 생산하는 brevicin 174Aγ의 서열과 상동성이 높은 KKKKVACTWG NAAAA로 분석되었다. brevicin 174Aγ 인접 유전자의 염기서열을 기반으로 프라이머를 설계하고 PCR 증폭 및 염기서열 분석을 수행한 결과, 2개의 박테리오신 유전자를 발견했다. 또한 N-말단 아미노산 서열은 구조적 breC 유전자의 아미노산 서열과 일치했다. 박테리오신 유전자 서열을 바탕으로 확인한 아미노산 서열은 brevicin 174Aγ의 아미노산 서열과 비교한 결과 breB 유전자에서는 신호 펩타이드의 단일 아미노산이 변한 반면, breC 유전자에서는 박테리오신 구조 유전자의 아미노산 3개가 다른 것이 확인되었다.

Acknowledgments

I would like to thank Editage (www.editage.co.kr) for English language editing.

Conflict of Interest

The authors have no conflict of interest to report.

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