- Email: [email protected]
Passive solar research and practice
Journal of Bioscience and Bioengineering VOL. 114 No. 5, 490e496, 2012 www.elsevier.com/locate/jbiosc
Monitoring of the multiple bacteriocin production by Enterococcus faecium NKR-5-3 through a developed liquid chromatography and mass spectrometry-based quantification system Rodney H. Perez,1 Kohei Himeno,1 Naoki Ishibashi,1 Yoshimitsu Masuda,1 Takeshi Zendo,1 Koji Fujita,1 Pongtep Wilaipun,2 Vichien Leelawatcharamas,3 Jiro Nakayama,1 and Kenji Sonomoto1, 4, * 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,1 Department of Fishery Products, Faculty of Fisheries, Kasetsart University, Chatuchak, Bangkok 10900, Thailand,2 Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok 10900, Thailand,3 and Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan4 Received 24 April 2012; accepted 5 June 2012 Available online 3 July 2012
Enterococcus faecium NKR-5-3 produces four antimicrobial peptides referred here as enterocins NKR-5-3A, B, C and D. A two-step electrospray ionization-liquid chromatography and mass spectrometry (ESI-LC/MS)-based quantification system was developed to monitor its multiple bacteriocin production profiles, which is essential in understanding the complex production regulation mechanism of strain NKR-5-3. The developed ESI-LC/MS-based quantification system can easily monitor the multiple bacteriocin production of this strain. Using the developed system, the production of enterocin NKR-5-3B was found to be not as variable as those of the other enterocins in different cultivation media. Production of enterocin NKR-5-3B was also found to have a wider optimum incubation temperature (20e30 C) than enterocins NKR-5-3A, C and D (25 C). Furthermore, at least 2 nM of the bacteriocin-like inducing peptide, enterocin NKR5-3D, regulated the production of NKR-5-3 enterocins except enterocin NKR-5-3B. These findings taken together suggest that enterocin NKR-5-3B has an independent production regulation mechanism from the other NKR-5-3 enterocins. The developed system could effectively pin-point the production profiles of the multiple bacteriocins of E. faecium NKR-5-3 under different fermentation conditions. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Lactic acid bacteria; Multiple bacteriocin; Bacteriocin quantification; Electrospray ionization-liquid chromatography and mass spectrometry (ESI-LC/MS); Induction]
Lactic acid bacteria (LAB) are ubiquitous in nature. They exist in various ecological niches including a wide array of fermented food products. LAB have a long history of application in fermented foods because of their beneficial influence on nutritional, organoleptic, and shelf-life on foods (1). Among the beneficial attributes of LAB, its ability to produce antimicrobial peptidesebacteriocins, has attracted particular attention both in food and pharmaceutical industries due to its potential use as natural food preservative and therapeutic antibiotics (2e4). Bacteriocins are ribosomally synthesized antibacterial peptides that possess antagonistic activity toward closely related strains, while its producer cells are immune to their own bacteriocins (1). Unlike common antibiotics, LAB bacteriocins are generally considered food-grade due to its typical association in food fermentation. The U.S. Food and Drug Administration (FDA) classified LAB and its metabolites as generally regarded as safe (GRAS) as human food ingredient (5). This distinction has given bacteriocin a critical advantage over common * Corresponding author at: 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. Tel./fax: þ81 92 642 3019. E-mail address: [email protected] (K. Sonomoto).
antibiotics in the legal stand point in its use in food applications. Moreover, the U.S. FDA’s approval of the use of nisin in pasteurized processed cheese spreads on 1988 has established a legal precedent in the U.S. for the use of bacteriocins as food preservative (4). However, in order to materialize the full potential of new bacteriocins as food preservatives, it is important first to understand the biology of these bacteriocins, and, in particular, to elucidate its structureefunction relationships, production, immunity, regulation and its mode of antimicrobial action (6). Enterococcus faecium NKR-5-3, isolated from a Thai fermented fish e Pla-ra, produces multiple bacteriocins, enterocins NKR-5-3A, Z, B, C and a bacteriocin-like inducing peptide, enterocin NKR-5-3D (7,8). Enterocin NKR-5-3A and Z forms a two-peptide bacteriocin that showed 100% and 95% homologies to the peptides of a known two-peptide bacteriocin brochocin-C (brochocins A and B respectively) (9), although enterocin NKR-5-3Z was not detected in the supernatant, its putative structure gene was found in the genome of strain NKR-5-3 (8). Enterocin NKR-5-3B is believed to be a novel bacteriocin, based on the fact that its molecular mass does not resemble to any reported bacteriocin, however its structure has not yet been elucidated since initial attempts to obtain its amino acid sequence by Edman Degradation were unsuccessful, indicating that
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.06.003
VOL. 114, 2012
MONITORING OF MULTIPLE BACTERIOCINS BY LC/MS
the access to the N-terminal residue was blocked (8). Enterocin NKR-5-3C is a novel bacteriocin belonging to class IIa bacteriocins (10). Enterocin NKR-5-3D on the other hand, is a bacteriocin-like inducing peptide that showed 89% homology to IP-TX, a known bacteriocin inducing peptide in Lactobacillus sakei 5 (11). However, unlike IP-TX, enterocin NKR-5-3D showed a weak antimicrobial activity (8) hence termed bacteriocin-like inducing peptide (12). It is believed that strain NKR-5-3 has a unique biosynthetic machinery that enables it to synthesize multiple bacteriocins without exhaustion of its energy. It is known that the regulation of bacteriocin production utilizes a quorum-sensing system which is either a two or three-component regulatory system. However, the biosynthesis of bacteriocins, in addition to the cell densitydependent genetic regulation (quorum-sensing) system, has also been shown to be influenced by fermentation conditions such as cultivation media, temperature, ionic strength and pH (13e16). Understanding the production regulation of these bacteriocins would be helpful in optimizing the productions, which would in turn facilitate in their potential applications. In addition, production regulation mechanism is one of the essential pieces to solving the bacteriocin biosynthetic mechanism puzzle. Therefore, a viable protocol that can monitor the production of each bacteriocin is essential. The conventional method of monitoring bacteriocin production, such as the spot-on-lawn antimicrobial assay (17), although fast and reliable, can only predict the total bacteriocin production rather than as individual bacteriocins. An immunoassay system that can monitor multiple bacteriocin production has been reported previously (14). However, they emphasized that the success in the generation of specific antibodies against each bacteriocin is highly dependent on the selection of antigenic fragments, carrier proteins, and conjugation sites and should be done with much care in accordance to the particularities for each bacteriocin, thus highlighting the tediousness of the system. On the other hand, the developmental integration of high performance liquid chromatography and mass spectrometry (LC/MS) has greatly helped in the discovery, evaluation and development of new drugs and other important biomolecules due to its inherent specificity, sensitivity, and speed in quantitative bio-analysis (18,19). However, its use on bacteriocin research is not as popular, despite the potential of bacteriocins as a new drug, targeting the multi-drug resistant pathogens. The use of LC/MS system in bacteriocin researches is focused on the detection and identification of novel bacteriocins rather than for quantification. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported for rapid detection and identification of bacteriocins from culture supernatant (20) and bacterial cells (21). Moreover, Zendo et al. (22) reported an electrospray ionization-liquid chromatography and mass spectrometry (ESI-LC/MS) protocol for rapid identification of novel bacteriocins. Ghobrial et al. (23) reports the first and only paper on the use of LC/MS as an analytical method for the quantification of lantibiotics in rat plasma. In this paper, we report the establishment and the use of an ESILC/MS-based quantification system that can monitor the production profiles of multiple bacteriocin production of strain NKR-5-3 as influenced by various fermentation conditions, which is essential in the understanding of its multiple bacteriocin production regulation. Understanding the production regulation profiles would in turn aid in the understanding of the complex biosynthetic mechanism of multiple bacteriocin production of strain NKR-5-3. MATERIALS AND METHODS Bacterial strains, media and stock solutions Strain NKR-5-3 was cultivated in M17 medium (Merck, Darmstadt, Germany) in most experiments and in Tryptic Soy Broth (Difco, Sparks, OK, USA) supplemented with 0.6% yeast extract (Nacalai Tesque, Kyoto, Japan) (TSBYE) for induction experiments. Indicator strains used in
491
this study were Enterococcus faecalis JCM 5803T, cultivated in MRS medium (Oxoid, Hampshire, UK), and Bacillus subtilis JCM 1465T, cultivated with agitation in TSBYE, at 37 C and 30 C respectively. All microorganisms were stored at 80 C in their respective media with 30% glycerol and cultivated twice before use. Stock solutions of NKR-5-3 enterocins were prepared by dissolving lyophilized purified bacteriocins in 10% (v/v) dimethyl sulfoxide (DMSO; Nacalai Tesque, Kyoto, Japan) and stored in low protein-binding tubes (Assist, Tokyo, Japan) at 30 C until use. Stock solution of the internal standard e nisin A (SigmaeAldrich, Steinheim, Germany) was also prepared using the same solvent after RP-HPLC purification and lyophilization as previously described (24). Concentrations of these stock solutions were quantified using BCA Protein Assay Kit Ô (Thermo Scientific, Rockford, IL, USA). ESI-LC/MS conditions The LC/MS analyses were performed using Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with JMS-T100LC ESI-TOF MS (JEOL, Tokyo, Japan). A styrene divinyl benzene copolymer analytical column (150 mm 2.1 mm; 5 mm particle size; Varian Inc., Shropshire, England) was used for chromatographic separation at 30 C. MilliQ water (solvent A) and acetonitrile (solvent B) with 0.05% (v/v) trifluoroacetic acid (TFA) were used as mobile phase at a flow rate of 0.2 ml/min in an optimized gradient, which is composed of 3-phases to facilitate continuous analyses: elution, column washing, and column equilibration. Elution phase ran a linear gradient of 25e60% (v/v) of solvent B for 35 min, followed by column washing for 10 min at 100% (v/v) of solvent B. Column equilibration phase was done for 15 min at 25% (v/v) of solvent B before 50 ml of sample was injected through automated sample injection system. The ESIMS system has a mass accuracy value of 5 ppm after calibration using a standard compound, reserpine, as per manufacturer’s instruction (JEOL). ESI-MS detection was performed from 10 to 40 min of elution program through an automatic valve switch. Ionizations were achieved through electrospray ionization in positive detection mode. Desolvation temperature was set at 260 C while needle, orifice, and ring lens voltages were set at 2000 V, 75 V and 10 V respectively. Other parameters were optimized, to provide highest sensitivity, by manual tuning using a purified nisin Z solution directly infused into the ESI source. MS data-dependent acquisition mode was set, in which MS spectra from m/z 500e3000 were collected and recorded at an interval of 2 ns. Data were acquired and processed using a JEOL MassCenter program (JEOL). Using this program, mass chromatograms corresponding to the dominant molecular species of each NKR-5-3 enterocin and the internal standard, as shown in Table 1, were extracted from the total ion chromatogram. Standard calibration curves Standard calibration curves were established by running the ESI-LC/MS program on standard solutions of NKR-5-3 enterocins. Standard solutions were prepared by doing two-fold serial dilutions of a working solution containing all NKR-5-3 enterocins at an initial concentration of 100 mg/ml. Internal standard was spiked to all standard solutions to a final concentration of 10 mg/ml prior to the analyses. The average of the peak area ratios of each NKR-5-3 enterocin to the internal standard from two independent runs was plotted as a function of NKR-5-3 enterocins concentration in the standard solutions. A regression equation of the calibration curve was used to calculate the concentrations of all the samples. The accuracy and sensitivity of the standard calibration curves were evaluated by quantifying the quality control (QC) samples using the regression line of the standard calibration curves. QC samples were prepared at three concentrations: low (1 mg/ml), medium (10 mg/ml), and high (100 mg/ml). Accuracy was expressed in percent (%) error in reference to the theoretical concentrations measured using BCA Protein Assay KitÔ. Sensitivity was assessed by determining the lowest quantifiable concentration (LQC). The LQC is the lowest concentration of the NKR-5-3 enterocins used in the calibration curve that still shows good fit to the regression line. Sample pre-treatment and bacteriocin quantification Samples were prepared by performing partial purification of the culture supernatant to eliminate the medium-derived impurities in the culture supernatant as well as to concentrate the bacteriocins. Various purification methods such as, hydrophobic interaction resin e Amberlite XAD-16 (SigmaeAldrich, Steinheim, Germany), ammonium sulfate ((NH4)2SO4) precipitation, disposable columns: Sep-PakÒ C18 and OASISÒ Plus HLB Cartridges (Waters, Milford, MA, USA), were evaluated for its effectiveness in partially purifying the peptides in the culture supernatant. Among the methods tested, the Amberlite XAD-16 method seemed the most appropriate in partially purifying the sample, especially since it is used in the previously reported TABLE 1. Multiple bacteriocins of E. faecium NKR-5-3 and ESI-LC/MS detection of bacteriocins including the internal standard e nisin A (purified from SigmaeAldrich stock).
Enterocin Enterocin Enterocin Enterocin Nisin A
NKR-5-3A NKR-5-3B NKR-5-3C NKR-5-3D
Molecular mass (Da)
Identification
Mass rangea (m/z)
Molecular ion charge
5242.3 6316.4 4512.8 2843.5 3354
Class IIb Novel bacteriocin Novel bacteriocin Inducing peptide Internal standard
1311.5e1312.5 1580e1581 1504e1506 948.5e949.5 1118.5e1119.5
[Mþ4H]4þ [Mþ4H]4þ [Mþ3H]3þ [Mþ3H]3þ [Mþ3H]3þ
a Dominant ion species that were used to detect target bacteriocins in the mass chromatogram.
PEREZ ET AL.
J. BIOSCI. BIOENG.,
Induction of bacteriocin production The induction assay was done by adding varying concentrations (0e50 nM) of chemically synthesized enterocin NKR5-3D at the early stage of growth (after 4 h) of strain NKR-5-3 cultivated in TSBYE medium for 24 h at 30 C. In this medium, the strain NKR-5-3 did not show any antibacterial activity against E. faecalis JCM 5803T (8). Bacteriocin productions were quantified using the developed quantification system described above. Spot-onlawn assays were also performed using E. faecalis JCM 5803T as the indicator strain.
RESULTS
Intensity
Total ion chromatogram
1000 0
B
Intensity
200
entNKR-5-3A m/z 1311.5 – 1312.5 (25.16 min)
100 0 1000
Intensity
Evaluation of the influence of fermentation conditions To evaluate the influence of cultivation media on the bacteriocin productions, the strain NKR-5-3 was cultivated in a nutritionally superior (M17) and nutritionally inferior (TSBYE) cultivation media whereas the influence of incubation temperature was investigated by cultivating the strain NKR-5-3 in M17 medium at different incubation temperatures using a water-bath incubator. Bacteriocin productions were determined using the developed method described above after cultivating the NKR-5-3 strain for 24 h. The conventional bacteriocin quantification assay: the spot-on-lawn method as described previously (8) was also performed to counter check the pattern of the results.
A
2000
C
entNKR-5-3B m/z 1580 – 1581
500
(36.83 min) 0
D
20 Intensity
purification scheme of the NKR-5-3 enterocins (8). Briefly, 2.5 g of activated Amberlite XAD-16 resins were soaked with agitation in 10 ml strain NKR-5-3 culture supernatant for 3 h and then washed thrice with 50% ethanol to remove impurities. Bacteriocins were eluted using 70% 2-propanol acidified with 0.1% (v/ v) TFA. The eluted fraction was then lyophilized to dryness and the resulting precipitate was dissolved in 1 ml 10% (v/v) DMSO, which served as the working solution. Internal standard was spiked to the working solution to a final concentration of 10 mg/ml prior to the ESI-LC/MS analysis as described above. NKR-5-3 enterocins were quantified using the established standard calibration curves specific to each bacteriocins. QC samples were included in every batch of experimental analysis to check the validity of the results.
entNKR-5-3C m/z 1504 – 1506 (26.53 min)
10 0
E
400 Intensity
492
entNKR-5-3D m/z 948.5 – 948.5 (29.06 min)
200 0
Sensitivity and accuracy of the quantification system The calibration curves for enterocins NKR-5-3A and C were linear over
F
100 Intensity
ESI-LC/MS-based quantification system The developed system is composed of a sample pre-treatment phase and the quantification phase. Partial purification using Amberlite XAD-16 of the samples was chosen as sample pre-treatment method as it effectively removes impurities, concentrates the bacteriocin without compromising recovery. Total bacteriocin activity from the supernatant was recovered after Amberlite XAD-16 purification (8). Moreover, the recoveries of spiked NKR-5-3 enterocins into uncultured M17 medium after Amberlite XAD-16 purification showed highly acceptable mean value of 97.38%. The quantification phase employs an optimized LC/MS-based standard calibration curves system. During the optimization of the LC/MS conditions, various chromatographic columns were screened for its effectiveness in separating the elution of each NKR5-3 peptide including the internal standard. Among the columns tested, VARIANÔ copolymer column effectively separated the elution of all bacteriocins. Enterocins NKR-5-3A, B, C, and D were eluted at approximately 25.16, 36.83, 26.53, and 29.06 min respectively, while the internal standard (nisin A) was eluted at approximately 22.62 min (Fig. 1AeF). Using other types of columns resulted in co-elution of some peptides specifically enterocins NKR5-3A and C. Analysis of the mass spectra of the affected chromatographic peaks showed that enterocins NKR-5-3A and C were co-eluted with each other (data not shown). Dominant molecular species of enterocins NKR-5-3A and B were in its quadruple-protonated molecular ion form [Mþ4H]4þ which were detected at the m/z range of 1311.5e1312.5 and 1580e1581 respectively. Minute amounts of triple and penta-protonated molecular species of both enterocins NKR-5-3A and B were also detected (data not shown). On the other hand, enterocins NKR-5-3C and D were dominant in its triple-protonated molecular ion form [Mþ3H]3þ and were detected at the m/z range of 1504e1506 and 948.5e949.5 respectively. Tiny signals of double and quadruple protons of both enterocins NKR-5-3C and D were also observed (data not shown). The internal standard was dominant as a tripleprotonated molecular ion and was detected at the m/z range of 1118.5e1119.5.
Internal standard (nisin A) m/z 1118.5 – 1119.5 (22.62 min)
50 0 0
10
20 Elution time (min)
30
40
FIG. 1. Mass chromatograms showing effective separation of the peptides using Varian Inc. copolymer column. Total ion chromatogram (A) and selected ion chromatograms of NKR-5-3 enterocins and the internal standard e nisin A (BeF) are shown. Respective elution times and dominant ion species (m/z) are indicated.
the range of 0.3906e100 mg/ml while for enterocins NKR-5-3B and D were linear at slightly higher concentration range of 0.7812e100 mg/ml. This linearity range shows the limit of quantifiable concentration of the calibration curves. The lower limit shows the LQC, hence referred as sensitivity limit. Furthermore, the correlation coefficients (R2) of the calibration curves were all greater than 0.993. The accuracy of the calibration curves of each NKR-5-3 enterocins was evaluated by quantifying three different concentrations of NKR-5-3 enterocin QC solutions. Measurements obtained from the developed method showed high accuracy expressed in % error in reference to the theoretical concentration. All measurements had less than 10% error, which falls within the acceptable margin of error in quantitative analysis. Moreover, statistical analysis shows that measurements have highly acceptable coefficient of variation which shows the precision of the system (Table 2). Influence of cultivation media on multiple bacteriocin production Cultivation media was found to influence the production of NKR-5-3 enterocins. It was previously reported that higher bacteriocin activity was observed when strain NKR-5-3 was cultivated in M17 medium than in TSBYE (8). When the production profiles of NKR-5-3 enterocins in M17 and TSBYE were monitored during the course of fermentation, it was surprising that bacteriocin production was detected earlier in nutritionally inferior TSBYE than in nutritionally superior M17 medium. After 4 h of incubation, bacteriocin production was detected in TSBYE
VOL. 114, 2012
MONITORING OF MULTIPLE BACTERIOCINS BY LC/MS
493
TABLE 2. Accuracy of the ESI-LC/MS-based calibration curves for each E. faecium NKR-5-3 peptide. ESI-LC/MS measured concentration (mg/ml)
a
Theoretical concentration
mg/ml (% R.S.D.)
EntNKR-5-3B (LQC ¼ 0.7812 mg/ml)
EntNKR-5-3A (LQC ¼ 0.3906 mg/ml)
EntNKR-5-3C (LQC ¼ 0.3906 mg/ml)
EntNKR-5-3D (LQC ¼ 0.7812 mg/ml)
Measured value
% error
Measured value
% error
Measured value
% error
Measured value
% error
1.10 9.14 93.83
10.0 8.6 6.17
0.98 10.38 99.12
2.0 3.8 0.88
0.98 9.47 103.07
2.0 5.3 3.07
1.10 10.16 104.22
10 1.6 4.22
1 (6.2) 10 (5.4) 100 (4.3)
% R.S.D. e Relative standard deviation 100. LQC: Lowest quantifiable concentration. a Measured using BCA Protein Assay Kit Ô (Thermo Scientific).
(Fig. 2A) while it is only after 6 h of incubation where bacteriocin production was detected in M17 medium (Fig. 2B). However, only enterocin NKR-5-3B and a small amount of enterocin NKR-5-3D (0.5 mg/ml which is approximately 180 nM) were detected in TSBYE medium (Fig. 2A) while all enterocins were detected simultaneously in M17 medium (Fig. 2B). More interestingly, the growth profiles showed that strain NKR-5-3 had longer lag phase in M17 than in TSBYE medium. It took approximately 4 h for the strain NKR-5-3 to establish itself in M17 while it only took approximately 2 h for it to adjust in TSBYE despite the fact that M17 is a more nutritionally complex medium than TSBYE. Nevertheless, the strain had better growth at the end of the fermentation in M17 than in TSBYE (Fig. 2C).
A
12
8
4 0 0
2
4
6
8
10 12 14 16 18 20
Incubation time (h)
B
12
8
4 0 0
2
4
6
8
10 12 14 16 18 20
Incubation time (h)
C
4
Cell density (OD600)
Induction of multiple bacteriocin production To elucidate the inducing activity of enterocin NKR-5-3D, different concentrations ranging from 0 to 50 nM (0.13 mg/ml) of chemically synthesized enterocin NKR-5-3D were added at the onset of log phase of growth (after 4 h) of strain NKR-5-3. Results obtained after 24-h cultivation showed that at least 2 nM (0.005 mg/ml) of enterocin NKR-5-3D is needed to induce the bacteriocin production (Fig. 4). This result was visible in both conventional antimicrobial assay and the developed ESI-LC/MS-based quantification system. However, the conventional assay can only estimate the influence of exogenous addition of enterocin NKR-5-3D to the total bacteriocin production (Fig. 4A) while the developed ESI-LC/MSbased system can pin-point its influence specific to each
Bacteriocin production (µg/ml)
Bacteriocin production (µg/ml)
Influence of temperature on multiple bacteriocin production Bacteriocin productions in strain NKR-5-3 were variable with incubation temperature with the exception of enterocin NKR-5-3B which showed a wide range of incubation temperature from 20 C to 30 C (Fig. 3A). Comparable productions of enterocin NKR-5-3B were observed between 20 C and 30 C of incubation temperature, whereas, the productions of the other NKR-5-3 enterocins were variable with temperature (Fig. 3A). This result is also visible using the conventional antimicrobial assay. Equal antibacterial activities were observed from 20 C to
30 C incubation temperatures against B. subtilis JCM 1465T (sensitive only to enterocin NKR-5-3B), whereas variable antimicrobial activity against E. faecalis JCM 5803T (sensitive to all NKR-5-3 enterocins) were observed from these incubation temperatures (Fig. 3B). Highest bacteriocin productivities were observed at 25 C despite comparable growth of the producer strain at 30 C. Furthermore, bacteriocin productions were significantly hampered beyond 30 C incubation temperature and bacteriocin productions were not detected at 40 C despite considerable cell growth (Fig. 3).
3 2 1 0 0
2
4
6
8 10 12 14 16 18 20 Incubation time (h)
FIG. 2. Growth and production profiles of multiple bacteriocins of E. faecium NKR-5-3. Bacteriocin productions in nutritionally simple (TSBYE) (A) and nutritionally complex (M17) cultivation media (B). Productions of NKR-5-3 enterocins are shown as follows: closed circles, entNKR-5-3A; closed triangles, entNKR-5-3B; closed squares, entNKR-5-3C; and open circles, entNKR-5-3D. Growth profiles of strain NKR-5-3 in these cultivation media (C); M17 (closed circles) and TSBYE (closed triangles) are shown.
494
PEREZ ET AL.
J. BIOSCI. BIOENG.,
FIG. 3. Influence of incubation temperature on the production of multiple bacteriocins of E. faecium NKR-5-3 in M17 medium after 24-h incubation. It was evaluated using the developed ESI-LC/MS-based quantification system (A) and using conventional antimicrobial assay (spot-on-lawn) (B). Productions of NKR-5-3 enterocins are shown as colored bars; black, entNKR-5-3A; dark gray, entNKR-5-3B; gray, entNKR-5-3C; light gray, entNKR-5-3D. Whereas bacteriocin activity against indicator strains are indicated as colored bars; black, B. subtilis JCM 1465T; and light gray, E. faecalis JCM 5803T. Growth of strain NKR-5-3 is indicated as closed circles.
enterocins. Using the developed method, it was found out that the enterocin NKR-5-3D cannot induce the production of enterocin NKR-5-3B (Fig. 4B). Interestingly, even without exogenous enterocin NKR-5-3D, strain NKR-5-3 in TSBYE medium can synthesize w180 nM (0.5 mg/ml) of enterocin NKR-5-3D but production of other NKR-5-3 enterocins were not induced (Fig. 2A).
DISCUSSION
3200
A
3
2 1600 1
0
0 0 0.5 1 2 3 4 5 10 50 EntNKR-5-3D concentration added (nM)
Cell density (OD600)
Bacteriocin activity (AU/ml)
E. faecium NKR-5-3 produces multiple bacteriocins including a bacteriocin-like production-inducing peptide (8). Some LAB strains have been reported to produce multiple bacteriocins (11,25). This is believed to be an important element in competition among bacteria since these bacteriocins have slightly different mode of actions which is important to the complementation of antimicrobial effects, thus widening the target cell spectrum and/or increasing the effectiveness of the target cell inhibition. It is also believed that multiple bacteriocin production can help the producer strain to abolish resistance problem of some target strains (26). In this paper, the regulation mechanism of multiple bacteriocin production of strain NKR-5-3 was studied by monitoring the production profiles of each enterocin as influenced by different fermentation conditions as well as influenced by its own bacteriocin-like inducing peptide. We developed and optimize a twostep ESI-LC/MS-based quantification system that can effectively monitor the production of each NKR-5-3 enterocin as influenced by various fermentation conditions.
Bacteriocin production (µg/ml)
Sample pre-treatment in LC/MS analysis is essential for the concentration of bacteriocins as well as the removal of mediumborne impurities (22). Moreover, proper separation of the elution of each bacteriocin is equally essential since co-elution would compromise the reliability of the measurements. The presence of co-eluting compounds could lead to under- (ion suppression) or over-estimation (ion enhancement) of the concentrations of the target molecule (27). Analysis of the mass chromatograms showed that impurity peaks were eluted before the elution of the target peptides (data not shown). Furthermore, columns other than copolymer-based columns resulted in co-elution of some NKR-5-3 enterocins, especially enterocins NKR-5-3 A and C (data not shown). Thus selection of the appropriate column for the analysis is very crucial in this developed system. In the case of NKR-5-3 enterocins, copolymer-based columns are effective in separating the elution of each peptide (Fig. 1). The conventional method for monitoring bacteriocin production, antimicrobial assays, can only predict the total bacteriocin productivity rather than individually. This limits the application of the conventional antimicrobial assay in monitoring multiple bacteriocin productions especially if the researcher wants to observe the production profile of the individual bacteriocins. Nevertheless, in cases where the bacteriocins have no overlapping antimicrobial activity, where an indicator microorganism is only sensitive to a single bacteriocin, the conventional antimicrobial assay has the advantage over the developed system. Unfortunately, the NKR-5-3 enterocins have overlapping antibacterial activity (8). The obvious advantages of the developed system over the conventional bacteriocin quantification antimicrobial assay are (i)
8
B
6 4 2 0 0
0.5
1
2
3
4
5
10
50
EntNKR-5-3D concentration added (nM)
FIG. 4. Induction of the production of multiple bacteriocins in TSBYE medium by one of its own peptide, enterocin NKR-5-3D. It was evaluated using conventional antimicrobial assay against E. faecalis JCM 5803T as indicator strain (spot-on-lawn) (A) and using the developed ESI-LC/MS-based quantification system (B). Vertical arrow indicates the threshold concentration (2 nM) of enterocin NKR-5-3D to induce bacteriocin production. Bacteriocin activity against the indicator strain is shown as closed circles while growth of strain NKR5-3 is shown as closed triangles. Whereas bacteriocin productions are indicated as follows: closed circles, entNKR-5-3A; closed triangles, entNKR-5-3B; closed squares, entNKR-53C; and open circles, entNKR-5-3D.
better data quality (visualizability), (ii) higher accuracy, and (iii) pin-points production profiles specific to each peptide. It has been demonstrated previously that fermentation conditions such as cultivation media and incubation temperature influence the bacteriocin production in strain NKR-5-3 (8). In TSBYE medium, only enterocin NKR-5-3B was detected at the onset on logarithmic growth phase (after 4 h) and minute amount of the inducing peptide during the later growth phase (Fig. 2A) while in M17 medium all enterocins were detected after 6 h of growth (Fig. 2B). Interestingly, the growth of strain NKR-5-3 in these media showed that it took longer time for the cells to establish itself in M17 than in TSBYE despite the fact that M17 medium is a more nutritious than TSBYE (Fig. 2C). This phenomenon does make biological sense since in M17 all bacteriocins were synthesized, the utilization of energy was focused on bacteriocin biosynthesis thereby compromising its growth. On the other hand, since in TSBYE only enterocin NKR-5-3B was synthesized at the early stage, more energy was available for growth. The much higher growth in M17 than in TSBYE at the end of fermentation supports this hypothesis (Fig. 2C). The synthesis of bacteriocin requires high amount of energy for both polymerization of building blocks (amino acids) and specific and coordinated control of bacteriocin gene expression (28). The high ATP requirement of bacteriocin synthesis further underlines its primary metabolite nature (29,30). Bacteriocin production in strain NKR-5-3 is dependent with incubation temperature (8). The optimal incubation temperature of NKR-5-3 peptides except enterocin NKR-5-3B, appeared to be at 25 C and productions are significantly reduced beyond 30 C. Enterocin NKR-5-3B appeared to have a wider range of incubation temperature from 20 C to 30 C (Fig. 3A). This phenomenon makes biological sense, since most bacteriocins display low potency at and above 37 C, especially the class IIa bacteriocins (15). A similar phenomenon was observed in a multiple bacteriocin producing strain E. faecium L50, where its bacteriocins have different optimal incubation temperatures. Maximum enterocins L50A and L50B production were detected at 25 C, while enterocins P and Q were maximally produced at 37 C and 47 C respectively (14). Strain NKR-5-3 produces a bacteriocin-like production-inducing peptide referred here as enterocin NKR-5-3D. The induction process activates after a threshold concentration of 2 nM of the inducing peptide is reach (Fig. 4). Similar threshold concentrations of the inducing peptides were reported in some LAB strains. Lactobacillus plantarum C11 requires at least 1 ng/ml (w5 nM) of the inducing peptide PlnA (12), while L. sakei LTH673 requires 0.2 ng/ml (w1 nM) of the inducing peptide IF for the induction mechanism to commence (31). However, in the case of L. sakei 5 which produces an inducing peptide IP-TX in which the inducing peptide of strain NKR-5-3, enterocin NKR-5-3D has 89% homology, requires a higher concentration of 400 ng/ml (w140 nM) to induce the bacteriocin production (11). However, in their study they investigated the induction of bacteriocin production at a high incubation temperature of 37 C, which is beyond the optimal condition for bacteriocin production. Enterocin NKR-5-3D induces the production of the strain NKR5-3 enterocins during the early growth phase and cannot induce its production once the cells already reached the stationary phase. Despite in the high concentration of enterocin NKR-5-3D, constant antibacterial activity was observed when it was added until 6 h (late log phase) of growth but antibacterial activity decreased tremendously when enterocin NKR-5-3D was added at 8 h (early stationary phase) (Fig. 5). This phenomenon makes sense since transcription process of genomic genes including those involved in three-component signal transducting system occurs during the early stage of growth (28). This finding is critical for the study of the regulation mechanism of the production of NKR-5-3 enterocins. In TSBYE medium, strain NKR-5-3 produces approximately 180 nM of
MONITORING OF MULTIPLE BACTERIOCINS BY LC/MS
495
12800
Bacteriocin activity (AU/ml)
VOL. 114, 2012
6400
0 Control
2
4
6
8
Induction time (h) FIG. 5. Growth-phase-dependency of the inducing activity of enterocin NKR-5-3D. Equal specific concentration (2.11 mM/OD600) of enterocin NKR-5-3D was added at different period of growth of E. faecium NKR-5-3 in TSBYE medium. Indicator microorganism used for the assay was E. faecalis JCM 5803T.
enterocin NKR-5-3D, which is more than enough concentration to induce the production of all its peptides yet it did not synthesize all the NKR-5-3 enterocins. Production of enterocin NKR-5-3D in TSBYE was only detected after 10 h of incubation (Fig. 2B). The growth-phase-dependency of the induction process in strain NKR5-3 may offer a logical explanation of this phenomenon. Similar phenomenon was observed by Brurberg et al. (32), their L. sake LTH673 strain loses the inducibility in the late logarithmic phase. They noted that activation of the auto-induction loop depends on the timely accumulation of the inducing peptide. In conclusion, this paper reports the establishment and use of an ESI-LC/MS-based quantification system that can pin-point the production of each NKR-5-3 enterocin as influenced by various fermentation conditions. To the best of our knowledge, this work is the first report on the use of ESI-LC/MS as a quantification tool in monitoring multiple bacteriocin production in lactic acid bacteria. This method proved to be very useful in the understanding the production regulation of the multiple bacteriocin production. ACKNOWLEDGMENTS This work was partially supported by the Ministry of Education, Culture, Sports, Science and Technology (Monbukagakusho) Scholarship Program of Japan, Grant-in-Aid for Scientific Research by the Japan Society for the Promotion of Science (JSPS), the JSPS-National Research Council of Thailand (NRCT) Core University Program on "Development of Thermotolerant Microbial Resources and Their Applications”, and the Research Grant for Young Investigators of Faculty of Agriculture, Kyushu University. References 1. de Vuyst, L. and Leroy, F.: Bacteriocins from lactic acid bacteria: production, purification, and food applications, J. Mol. Microbiol. Biotechnol., 13, 194e199 (2007). 2. Cleveland, J., Montville, T. J., Nes, I. F., and Chikindas, M. L.: Bacteriocins: safe, natural antimicrobials for food preservation, Int. J. Food Microbiol., 71, 1e20 (2001). 3. Riley, M. A. and Wertz, J. E.: Bacteriocins: evolution, ecology, and application, Annu. Rev. Microbiol., 56, 117e137 (2002). 4. Chen, H. and Hoover, D. G.: Bacteriocins and their food applications, Compr. Rev. Food Sci. Food Saf., 2, 82e100 (2003). 5. U.S. Federal Register (U.S. Food and Drug Administration): Nisin preparation: affirmation of GRAS status as a direct human food ingredient. 21 CFR Part 184, Fed. Reg., 53, 11247e11251 (1988). 6. Cotter, P. D., Hill, C., and Ross, R. P.: Bacteriocins: developing innate immunity for food, Nat. Rev. Microbiol., 3, 777e788 (2005).
496
PEREZ ET AL.
7. Wilaipun, P., Zendo, T., Sangjindavong, M., Nitisinprasert, S., Leelawatcharamas, V., Nakayama, J., and Sonomoto, K.: The two-synergistic peptide bacteriocin produced by Enterococcus faecium NKR-5-3 isolated from Thai fermented fish (Pla-ra), Sci. Asia, 30, 115e122 (2004). 8. Ishibashi, N., Himeno, K., Fujita, K., Masuda, Y., Perez, R. H., Zendo, T., Wilaipun, P., Leelawatcharamas, V., Nakayama, J., and Sonomoto K.: Purification and characterization of multiple bacteriocins and an inducing peptide produced by Enterococcus faecium NKR-5-3 from Thai fermented fish. Biosci. Biotechnol. Biochem, 76, 947e953 (2012). 9. Siragusa, G. R. and Cutter, C. N.: Brochocin-C, a new bacteriocin produced by Brochotrix campestris, Appl. Environ. Microbiol., 59, 2326e2328 (1993). 10. Himeno, K., Fujita, K., Zendo, T., Wilaipun, P., Ishibashi, N., Masuda, Y., Yoneyama, F., Leelawatcharamas, V., Nakayama, J., and Sonomoto, K.: Identification of enterocin NKR-5-3C, a novel class IIa bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3. Biosci. Biotechnol. Biochem, 76, 1245e1247 (2012). 11. Vaughan, A., Eijsink, V. G., and van Sinderen, D.: Functional characterization of a composite bacteriocin locus from malt isolate Lactobacillus sakei 5, Appl. Environ. Microbiol., 69, 7194e7203 (2003). 12. Diep, D. B., Håvarstein, L. S., and Nes, I. F.: A bacteriocin-like peptides induces bacteriocin synthesis in Lactobacillus plantarum C11, Mol. Microbiol., 18, 631e639 (1995). 13. Diep, D. B., Axelsson, L., Grefsli, C., and Nes, I. F.: The synthesis of the bacteriocin sakacin A is a temperature-sensitive process regulated by a pheromone peptide through a three-component regulatory system, Microbiology, 146, 2155e2160 (2000). 14. Criado, R., Gutierrez, J., Martin, M., Herranz, C., Hernandez, P. E., and Cintas, L. M.: Immunological characterization of temperature-regulated production of enterocin L50 (EntL50A and EntL50), enterocin P, and enterocin Q by Enterococcus faecium L50, Appl. Environ. Microbiol., 72, 7634e7643 (2006). 15. Fimland, G., Johnsen, L., Axelsson, L., Brurberg, M. B., Nes, I. F., Eijsink, V. G., and Meyer, J. N.: A C-terminal disulfide bridge in pediocin-like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum, J. Bacteriol., 182, 2643e2648 (2000). 16. Leroy, F. and de Vuyst, L.: The presence of salt and a curing agent reduces bacteriocin production by Lactobacillus sakei CTC 494, a potential starter culture for sausage fermentation, Appl. Environ. Microbiol., 65, 5350e5356 (1999). 17. Vera Pingitore, E., Salvucci, E., Sesma, F., and Nader-Macías, M. E.: Different strategies for purification of antimicrobial peptides from lactic acid bacteria (LAB), pp. 557e568, in: Mendez-Vilas, A. (Ed.), Communicating current research and educational topics and trends in applied microbiology. Microbiology book series, vol. 2. Formatex, Spain (2007). 18. Korfmacher, W. A.: Foundation review: principles and applications of LC-MS in new drug discovery, Drug Discov. Today Rev., 10, 1357e1367 (2005).
J. BIOSCI. BIOENG., 19. Xu, R. N., Fan, L., Rieser, M. J., and El-Shourbagy, T. A.: Recent advances in high-throughput quantitative bioanalysis by LC-MS/MS, J. Pharmaceut. Biomed., 44, 342e355 (2007). 20. Rose, N. L., Sporns, P., and McMullen, L. M.: Detection of bacteriocins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Appl. Environ. Microbiol., 65, 2238e2242 (1999). 21. Hindré, T., Didelot, S., Le Pennec, J. P., Haras, D., Dufour, A., and ValleeRehel, K.: Bacteriocin detection from whole bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry, Appl. Environ. Microbiol., 69, 1051e1058 (2003). 22. Zendo, T., Nakayama, J., Fujita, K., and Sonomoto, K.: Bacteriocin detection by liquid chromatography/mass spectrometry for rapid identification, J. Appl. Microbiol., 104, 499e507 (2008). 23. Ghobrial, O. G., Derendorf, H., and Hillman, J. D.: Development and validation of a LC-MS quantification method for the lantibiotic MU1140 in rat plasma, J. Pharm. Biomed. Anal., 49, 970e975 (2009). 24. Fujita, K., Ichimasa, S., Zendo, T., Koga, S., Yoneyama, F., Nakayama, J., and Sonomoto, K.: Structural analysis and characterization of lacticin Q, a novel bacteriocin belonging to a new family of unmodified bacteriocins of grampositive bacteria, Appl. Environ. Microbiol., 9, 2871e2877 (2007). 25. Cintas, L. M., Casaus, P., Herranz, C., Håvarstein, L. S., Hernandez, P. E., and Nes, I. F.: Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q, J. Bacteriol., 182, 6806e6814 (2000). 26. Eijsink, V. G., Axelsson, L., Diep, D. B., Håvarstein, L. S., Holo, H., and Nes, I. F.: Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication, Antonie van Leeuwenhoek, 81, 639e654 (2002). 27. Annesley, T. M.: Ion suppression in mass spectrometry, Clin. Chem. Min. Rev., 49, 1041e1044 (2003). 28. de Vuyst, L., Callewaert, R., and Crabbé, K.: Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavorable growth conditions, Microbiology, 142, 817e827 (1996). 29. Martin, S. A. and Russell, J. B.: Transport and phosphorylation of disaccharides by ruminal bacterium Streptococcus bovis, Appl. Environ. Microbiol., 53, 2388e2393 (1977). 30. Mataragas, M., Drosinos, E. H., Tsakalidou, E., and Metaxopoulos, J.: Influence of nutrients on growth and bacteriocin production by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, Antonie van Leeuwenhoek, 85, 191e198 (2004). 31. Eijsink, V. G., Brurberg, M. B., Middlehoven, P. H., and Nes, I. F.: Induction of bacteriocin production in Lactobacillus sakei by a secreted peptide, J. Bacteriol., 178, 2232e2237 (1996). 32. Brurberg, M. B., Nes, I. F., and Eijsink, G. H.: Pheromone-induced production of antimicrobial peptides in Lactobacillus, Mol. Microbiol., 26, 347e360 (1997).
Monitoring of the multiple bacteriocin production by Enterococcus faecium NKR-5-3 through a developed liquid chromatography and mass spectrometry-based quantification system Rodney H. Perez,1 Kohei Himeno,1 Naoki Ishibashi,1 Yoshimitsu Masuda,1 Takeshi Zendo,1 Koji Fujita,1 Pongtep Wilaipun,2 Vichien Leelawatcharamas,3 Jiro Nakayama,1 and Kenji Sonomoto1, 4, * 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,1 Department of Fishery Products, Faculty of Fisheries, Kasetsart University, Chatuchak, Bangkok 10900, Thailand,2 Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok 10900, Thailand,3 and Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan4 Received 24 April 2012; accepted 5 June 2012 Available online 3 July 2012
Enterococcus faecium NKR-5-3 produces four antimicrobial peptides referred here as enterocins NKR-5-3A, B, C and D. A two-step electrospray ionization-liquid chromatography and mass spectrometry (ESI-LC/MS)-based quantification system was developed to monitor its multiple bacteriocin production profiles, which is essential in understanding the complex production regulation mechanism of strain NKR-5-3. The developed ESI-LC/MS-based quantification system can easily monitor the multiple bacteriocin production of this strain. Using the developed system, the production of enterocin NKR-5-3B was found to be not as variable as those of the other enterocins in different cultivation media. Production of enterocin NKR-5-3B was also found to have a wider optimum incubation temperature (20e30 C) than enterocins NKR-5-3A, C and D (25 C). Furthermore, at least 2 nM of the bacteriocin-like inducing peptide, enterocin NKR5-3D, regulated the production of NKR-5-3 enterocins except enterocin NKR-5-3B. These findings taken together suggest that enterocin NKR-5-3B has an independent production regulation mechanism from the other NKR-5-3 enterocins. The developed system could effectively pin-point the production profiles of the multiple bacteriocins of E. faecium NKR-5-3 under different fermentation conditions. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Lactic acid bacteria; Multiple bacteriocin; Bacteriocin quantification; Electrospray ionization-liquid chromatography and mass spectrometry (ESI-LC/MS); Induction]
Lactic acid bacteria (LAB) are ubiquitous in nature. They exist in various ecological niches including a wide array of fermented food products. LAB have a long history of application in fermented foods because of their beneficial influence on nutritional, organoleptic, and shelf-life on foods (1). Among the beneficial attributes of LAB, its ability to produce antimicrobial peptidesebacteriocins, has attracted particular attention both in food and pharmaceutical industries due to its potential use as natural food preservative and therapeutic antibiotics (2e4). Bacteriocins are ribosomally synthesized antibacterial peptides that possess antagonistic activity toward closely related strains, while its producer cells are immune to their own bacteriocins (1). Unlike common antibiotics, LAB bacteriocins are generally considered food-grade due to its typical association in food fermentation. The U.S. Food and Drug Administration (FDA) classified LAB and its metabolites as generally regarded as safe (GRAS) as human food ingredient (5). This distinction has given bacteriocin a critical advantage over common * Corresponding author at: 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. Tel./fax: þ81 92 642 3019. E-mail address: [email protected] (K. Sonomoto).
antibiotics in the legal stand point in its use in food applications. Moreover, the U.S. FDA’s approval of the use of nisin in pasteurized processed cheese spreads on 1988 has established a legal precedent in the U.S. for the use of bacteriocins as food preservative (4). However, in order to materialize the full potential of new bacteriocins as food preservatives, it is important first to understand the biology of these bacteriocins, and, in particular, to elucidate its structureefunction relationships, production, immunity, regulation and its mode of antimicrobial action (6). Enterococcus faecium NKR-5-3, isolated from a Thai fermented fish e Pla-ra, produces multiple bacteriocins, enterocins NKR-5-3A, Z, B, C and a bacteriocin-like inducing peptide, enterocin NKR-5-3D (7,8). Enterocin NKR-5-3A and Z forms a two-peptide bacteriocin that showed 100% and 95% homologies to the peptides of a known two-peptide bacteriocin brochocin-C (brochocins A and B respectively) (9), although enterocin NKR-5-3Z was not detected in the supernatant, its putative structure gene was found in the genome of strain NKR-5-3 (8). Enterocin NKR-5-3B is believed to be a novel bacteriocin, based on the fact that its molecular mass does not resemble to any reported bacteriocin, however its structure has not yet been elucidated since initial attempts to obtain its amino acid sequence by Edman Degradation were unsuccessful, indicating that
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.06.003
VOL. 114, 2012
MONITORING OF MULTIPLE BACTERIOCINS BY LC/MS
the access to the N-terminal residue was blocked (8). Enterocin NKR-5-3C is a novel bacteriocin belonging to class IIa bacteriocins (10). Enterocin NKR-5-3D on the other hand, is a bacteriocin-like inducing peptide that showed 89% homology to IP-TX, a known bacteriocin inducing peptide in Lactobacillus sakei 5 (11). However, unlike IP-TX, enterocin NKR-5-3D showed a weak antimicrobial activity (8) hence termed bacteriocin-like inducing peptide (12). It is believed that strain NKR-5-3 has a unique biosynthetic machinery that enables it to synthesize multiple bacteriocins without exhaustion of its energy. It is known that the regulation of bacteriocin production utilizes a quorum-sensing system which is either a two or three-component regulatory system. However, the biosynthesis of bacteriocins, in addition to the cell densitydependent genetic regulation (quorum-sensing) system, has also been shown to be influenced by fermentation conditions such as cultivation media, temperature, ionic strength and pH (13e16). Understanding the production regulation of these bacteriocins would be helpful in optimizing the productions, which would in turn facilitate in their potential applications. In addition, production regulation mechanism is one of the essential pieces to solving the bacteriocin biosynthetic mechanism puzzle. Therefore, a viable protocol that can monitor the production of each bacteriocin is essential. The conventional method of monitoring bacteriocin production, such as the spot-on-lawn antimicrobial assay (17), although fast and reliable, can only predict the total bacteriocin production rather than as individual bacteriocins. An immunoassay system that can monitor multiple bacteriocin production has been reported previously (14). However, they emphasized that the success in the generation of specific antibodies against each bacteriocin is highly dependent on the selection of antigenic fragments, carrier proteins, and conjugation sites and should be done with much care in accordance to the particularities for each bacteriocin, thus highlighting the tediousness of the system. On the other hand, the developmental integration of high performance liquid chromatography and mass spectrometry (LC/MS) has greatly helped in the discovery, evaluation and development of new drugs and other important biomolecules due to its inherent specificity, sensitivity, and speed in quantitative bio-analysis (18,19). However, its use on bacteriocin research is not as popular, despite the potential of bacteriocins as a new drug, targeting the multi-drug resistant pathogens. The use of LC/MS system in bacteriocin researches is focused on the detection and identification of novel bacteriocins rather than for quantification. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported for rapid detection and identification of bacteriocins from culture supernatant (20) and bacterial cells (21). Moreover, Zendo et al. (22) reported an electrospray ionization-liquid chromatography and mass spectrometry (ESI-LC/MS) protocol for rapid identification of novel bacteriocins. Ghobrial et al. (23) reports the first and only paper on the use of LC/MS as an analytical method for the quantification of lantibiotics in rat plasma. In this paper, we report the establishment and the use of an ESILC/MS-based quantification system that can monitor the production profiles of multiple bacteriocin production of strain NKR-5-3 as influenced by various fermentation conditions, which is essential in the understanding of its multiple bacteriocin production regulation. Understanding the production regulation profiles would in turn aid in the understanding of the complex biosynthetic mechanism of multiple bacteriocin production of strain NKR-5-3. MATERIALS AND METHODS Bacterial strains, media and stock solutions Strain NKR-5-3 was cultivated in M17 medium (Merck, Darmstadt, Germany) in most experiments and in Tryptic Soy Broth (Difco, Sparks, OK, USA) supplemented with 0.6% yeast extract (Nacalai Tesque, Kyoto, Japan) (TSBYE) for induction experiments. Indicator strains used in
491
this study were Enterococcus faecalis JCM 5803T, cultivated in MRS medium (Oxoid, Hampshire, UK), and Bacillus subtilis JCM 1465T, cultivated with agitation in TSBYE, at 37 C and 30 C respectively. All microorganisms were stored at 80 C in their respective media with 30% glycerol and cultivated twice before use. Stock solutions of NKR-5-3 enterocins were prepared by dissolving lyophilized purified bacteriocins in 10% (v/v) dimethyl sulfoxide (DMSO; Nacalai Tesque, Kyoto, Japan) and stored in low protein-binding tubes (Assist, Tokyo, Japan) at 30 C until use. Stock solution of the internal standard e nisin A (SigmaeAldrich, Steinheim, Germany) was also prepared using the same solvent after RP-HPLC purification and lyophilization as previously described (24). Concentrations of these stock solutions were quantified using BCA Protein Assay Kit Ô (Thermo Scientific, Rockford, IL, USA). ESI-LC/MS conditions The LC/MS analyses were performed using Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with JMS-T100LC ESI-TOF MS (JEOL, Tokyo, Japan). A styrene divinyl benzene copolymer analytical column (150 mm 2.1 mm; 5 mm particle size; Varian Inc., Shropshire, England) was used for chromatographic separation at 30 C. MilliQ water (solvent A) and acetonitrile (solvent B) with 0.05% (v/v) trifluoroacetic acid (TFA) were used as mobile phase at a flow rate of 0.2 ml/min in an optimized gradient, which is composed of 3-phases to facilitate continuous analyses: elution, column washing, and column equilibration. Elution phase ran a linear gradient of 25e60% (v/v) of solvent B for 35 min, followed by column washing for 10 min at 100% (v/v) of solvent B. Column equilibration phase was done for 15 min at 25% (v/v) of solvent B before 50 ml of sample was injected through automated sample injection system. The ESIMS system has a mass accuracy value of 5 ppm after calibration using a standard compound, reserpine, as per manufacturer’s instruction (JEOL). ESI-MS detection was performed from 10 to 40 min of elution program through an automatic valve switch. Ionizations were achieved through electrospray ionization in positive detection mode. Desolvation temperature was set at 260 C while needle, orifice, and ring lens voltages were set at 2000 V, 75 V and 10 V respectively. Other parameters were optimized, to provide highest sensitivity, by manual tuning using a purified nisin Z solution directly infused into the ESI source. MS data-dependent acquisition mode was set, in which MS spectra from m/z 500e3000 were collected and recorded at an interval of 2 ns. Data were acquired and processed using a JEOL MassCenter program (JEOL). Using this program, mass chromatograms corresponding to the dominant molecular species of each NKR-5-3 enterocin and the internal standard, as shown in Table 1, were extracted from the total ion chromatogram. Standard calibration curves Standard calibration curves were established by running the ESI-LC/MS program on standard solutions of NKR-5-3 enterocins. Standard solutions were prepared by doing two-fold serial dilutions of a working solution containing all NKR-5-3 enterocins at an initial concentration of 100 mg/ml. Internal standard was spiked to all standard solutions to a final concentration of 10 mg/ml prior to the analyses. The average of the peak area ratios of each NKR-5-3 enterocin to the internal standard from two independent runs was plotted as a function of NKR-5-3 enterocins concentration in the standard solutions. A regression equation of the calibration curve was used to calculate the concentrations of all the samples. The accuracy and sensitivity of the standard calibration curves were evaluated by quantifying the quality control (QC) samples using the regression line of the standard calibration curves. QC samples were prepared at three concentrations: low (1 mg/ml), medium (10 mg/ml), and high (100 mg/ml). Accuracy was expressed in percent (%) error in reference to the theoretical concentrations measured using BCA Protein Assay KitÔ. Sensitivity was assessed by determining the lowest quantifiable concentration (LQC). The LQC is the lowest concentration of the NKR-5-3 enterocins used in the calibration curve that still shows good fit to the regression line. Sample pre-treatment and bacteriocin quantification Samples were prepared by performing partial purification of the culture supernatant to eliminate the medium-derived impurities in the culture supernatant as well as to concentrate the bacteriocins. Various purification methods such as, hydrophobic interaction resin e Amberlite XAD-16 (SigmaeAldrich, Steinheim, Germany), ammonium sulfate ((NH4)2SO4) precipitation, disposable columns: Sep-PakÒ C18 and OASISÒ Plus HLB Cartridges (Waters, Milford, MA, USA), were evaluated for its effectiveness in partially purifying the peptides in the culture supernatant. Among the methods tested, the Amberlite XAD-16 method seemed the most appropriate in partially purifying the sample, especially since it is used in the previously reported TABLE 1. Multiple bacteriocins of E. faecium NKR-5-3 and ESI-LC/MS detection of bacteriocins including the internal standard e nisin A (purified from SigmaeAldrich stock).
Enterocin Enterocin Enterocin Enterocin Nisin A
NKR-5-3A NKR-5-3B NKR-5-3C NKR-5-3D
Molecular mass (Da)
Identification
Mass rangea (m/z)
Molecular ion charge
5242.3 6316.4 4512.8 2843.5 3354
Class IIb Novel bacteriocin Novel bacteriocin Inducing peptide Internal standard
1311.5e1312.5 1580e1581 1504e1506 948.5e949.5 1118.5e1119.5
[Mþ4H]4þ [Mþ4H]4þ [Mþ3H]3þ [Mþ3H]3þ [Mþ3H]3þ
a Dominant ion species that were used to detect target bacteriocins in the mass chromatogram.
PEREZ ET AL.
J. BIOSCI. BIOENG.,
Induction of bacteriocin production The induction assay was done by adding varying concentrations (0e50 nM) of chemically synthesized enterocin NKR5-3D at the early stage of growth (after 4 h) of strain NKR-5-3 cultivated in TSBYE medium for 24 h at 30 C. In this medium, the strain NKR-5-3 did not show any antibacterial activity against E. faecalis JCM 5803T (8). Bacteriocin productions were quantified using the developed quantification system described above. Spot-onlawn assays were also performed using E. faecalis JCM 5803T as the indicator strain.
RESULTS
Intensity
Total ion chromatogram
1000 0
B
Intensity
200
entNKR-5-3A m/z 1311.5 – 1312.5 (25.16 min)
100 0 1000
Intensity
Evaluation of the influence of fermentation conditions To evaluate the influence of cultivation media on the bacteriocin productions, the strain NKR-5-3 was cultivated in a nutritionally superior (M17) and nutritionally inferior (TSBYE) cultivation media whereas the influence of incubation temperature was investigated by cultivating the strain NKR-5-3 in M17 medium at different incubation temperatures using a water-bath incubator. Bacteriocin productions were determined using the developed method described above after cultivating the NKR-5-3 strain for 24 h. The conventional bacteriocin quantification assay: the spot-on-lawn method as described previously (8) was also performed to counter check the pattern of the results.
A
2000
C
entNKR-5-3B m/z 1580 – 1581
500
(36.83 min) 0
D
20 Intensity
purification scheme of the NKR-5-3 enterocins (8). Briefly, 2.5 g of activated Amberlite XAD-16 resins were soaked with agitation in 10 ml strain NKR-5-3 culture supernatant for 3 h and then washed thrice with 50% ethanol to remove impurities. Bacteriocins were eluted using 70% 2-propanol acidified with 0.1% (v/ v) TFA. The eluted fraction was then lyophilized to dryness and the resulting precipitate was dissolved in 1 ml 10% (v/v) DMSO, which served as the working solution. Internal standard was spiked to the working solution to a final concentration of 10 mg/ml prior to the ESI-LC/MS analysis as described above. NKR-5-3 enterocins were quantified using the established standard calibration curves specific to each bacteriocins. QC samples were included in every batch of experimental analysis to check the validity of the results.
entNKR-5-3C m/z 1504 – 1506 (26.53 min)
10 0
E
400 Intensity
492
entNKR-5-3D m/z 948.5 – 948.5 (29.06 min)
200 0
Sensitivity and accuracy of the quantification system The calibration curves for enterocins NKR-5-3A and C were linear over
F
100 Intensity
ESI-LC/MS-based quantification system The developed system is composed of a sample pre-treatment phase and the quantification phase. Partial purification using Amberlite XAD-16 of the samples was chosen as sample pre-treatment method as it effectively removes impurities, concentrates the bacteriocin without compromising recovery. Total bacteriocin activity from the supernatant was recovered after Amberlite XAD-16 purification (8). Moreover, the recoveries of spiked NKR-5-3 enterocins into uncultured M17 medium after Amberlite XAD-16 purification showed highly acceptable mean value of 97.38%. The quantification phase employs an optimized LC/MS-based standard calibration curves system. During the optimization of the LC/MS conditions, various chromatographic columns were screened for its effectiveness in separating the elution of each NKR5-3 peptide including the internal standard. Among the columns tested, VARIANÔ copolymer column effectively separated the elution of all bacteriocins. Enterocins NKR-5-3A, B, C, and D were eluted at approximately 25.16, 36.83, 26.53, and 29.06 min respectively, while the internal standard (nisin A) was eluted at approximately 22.62 min (Fig. 1AeF). Using other types of columns resulted in co-elution of some peptides specifically enterocins NKR5-3A and C. Analysis of the mass spectra of the affected chromatographic peaks showed that enterocins NKR-5-3A and C were co-eluted with each other (data not shown). Dominant molecular species of enterocins NKR-5-3A and B were in its quadruple-protonated molecular ion form [Mþ4H]4þ which were detected at the m/z range of 1311.5e1312.5 and 1580e1581 respectively. Minute amounts of triple and penta-protonated molecular species of both enterocins NKR-5-3A and B were also detected (data not shown). On the other hand, enterocins NKR-5-3C and D were dominant in its triple-protonated molecular ion form [Mþ3H]3þ and were detected at the m/z range of 1504e1506 and 948.5e949.5 respectively. Tiny signals of double and quadruple protons of both enterocins NKR-5-3C and D were also observed (data not shown). The internal standard was dominant as a tripleprotonated molecular ion and was detected at the m/z range of 1118.5e1119.5.
Internal standard (nisin A) m/z 1118.5 – 1119.5 (22.62 min)
50 0 0
10
20 Elution time (min)
30
40
FIG. 1. Mass chromatograms showing effective separation of the peptides using Varian Inc. copolymer column. Total ion chromatogram (A) and selected ion chromatograms of NKR-5-3 enterocins and the internal standard e nisin A (BeF) are shown. Respective elution times and dominant ion species (m/z) are indicated.
the range of 0.3906e100 mg/ml while for enterocins NKR-5-3B and D were linear at slightly higher concentration range of 0.7812e100 mg/ml. This linearity range shows the limit of quantifiable concentration of the calibration curves. The lower limit shows the LQC, hence referred as sensitivity limit. Furthermore, the correlation coefficients (R2) of the calibration curves were all greater than 0.993. The accuracy of the calibration curves of each NKR-5-3 enterocins was evaluated by quantifying three different concentrations of NKR-5-3 enterocin QC solutions. Measurements obtained from the developed method showed high accuracy expressed in % error in reference to the theoretical concentration. All measurements had less than 10% error, which falls within the acceptable margin of error in quantitative analysis. Moreover, statistical analysis shows that measurements have highly acceptable coefficient of variation which shows the precision of the system (Table 2). Influence of cultivation media on multiple bacteriocin production Cultivation media was found to influence the production of NKR-5-3 enterocins. It was previously reported that higher bacteriocin activity was observed when strain NKR-5-3 was cultivated in M17 medium than in TSBYE (8). When the production profiles of NKR-5-3 enterocins in M17 and TSBYE were monitored during the course of fermentation, it was surprising that bacteriocin production was detected earlier in nutritionally inferior TSBYE than in nutritionally superior M17 medium. After 4 h of incubation, bacteriocin production was detected in TSBYE
VOL. 114, 2012
MONITORING OF MULTIPLE BACTERIOCINS BY LC/MS
493
TABLE 2. Accuracy of the ESI-LC/MS-based calibration curves for each E. faecium NKR-5-3 peptide. ESI-LC/MS measured concentration (mg/ml)
a
Theoretical concentration
mg/ml (% R.S.D.)
EntNKR-5-3B (LQC ¼ 0.7812 mg/ml)
EntNKR-5-3A (LQC ¼ 0.3906 mg/ml)
EntNKR-5-3C (LQC ¼ 0.3906 mg/ml)
EntNKR-5-3D (LQC ¼ 0.7812 mg/ml)
Measured value
% error
Measured value
% error
Measured value
% error
Measured value
% error
1.10 9.14 93.83
10.0 8.6 6.17
0.98 10.38 99.12
2.0 3.8 0.88
0.98 9.47 103.07
2.0 5.3 3.07
1.10 10.16 104.22
10 1.6 4.22
1 (6.2) 10 (5.4) 100 (4.3)
% R.S.D. e Relative standard deviation 100. LQC: Lowest quantifiable concentration. a Measured using BCA Protein Assay Kit Ô (Thermo Scientific).
(Fig. 2A) while it is only after 6 h of incubation where bacteriocin production was detected in M17 medium (Fig. 2B). However, only enterocin NKR-5-3B and a small amount of enterocin NKR-5-3D (0.5 mg/ml which is approximately 180 nM) were detected in TSBYE medium (Fig. 2A) while all enterocins were detected simultaneously in M17 medium (Fig. 2B). More interestingly, the growth profiles showed that strain NKR-5-3 had longer lag phase in M17 than in TSBYE medium. It took approximately 4 h for the strain NKR-5-3 to establish itself in M17 while it only took approximately 2 h for it to adjust in TSBYE despite the fact that M17 is a more nutritionally complex medium than TSBYE. Nevertheless, the strain had better growth at the end of the fermentation in M17 than in TSBYE (Fig. 2C).
A
12
8
4 0 0
2
4
6
8
10 12 14 16 18 20
Incubation time (h)
B
12
8
4 0 0
2
4
6
8
10 12 14 16 18 20
Incubation time (h)
C
4
Cell density (OD600)
Induction of multiple bacteriocin production To elucidate the inducing activity of enterocin NKR-5-3D, different concentrations ranging from 0 to 50 nM (0.13 mg/ml) of chemically synthesized enterocin NKR-5-3D were added at the onset of log phase of growth (after 4 h) of strain NKR-5-3. Results obtained after 24-h cultivation showed that at least 2 nM (0.005 mg/ml) of enterocin NKR-5-3D is needed to induce the bacteriocin production (Fig. 4). This result was visible in both conventional antimicrobial assay and the developed ESI-LC/MS-based quantification system. However, the conventional assay can only estimate the influence of exogenous addition of enterocin NKR-5-3D to the total bacteriocin production (Fig. 4A) while the developed ESI-LC/MSbased system can pin-point its influence specific to each
Bacteriocin production (µg/ml)
Bacteriocin production (µg/ml)
Influence of temperature on multiple bacteriocin production Bacteriocin productions in strain NKR-5-3 were variable with incubation temperature with the exception of enterocin NKR-5-3B which showed a wide range of incubation temperature from 20 C to 30 C (Fig. 3A). Comparable productions of enterocin NKR-5-3B were observed between 20 C and 30 C of incubation temperature, whereas, the productions of the other NKR-5-3 enterocins were variable with temperature (Fig. 3A). This result is also visible using the conventional antimicrobial assay. Equal antibacterial activities were observed from 20 C to
30 C incubation temperatures against B. subtilis JCM 1465T (sensitive only to enterocin NKR-5-3B), whereas variable antimicrobial activity against E. faecalis JCM 5803T (sensitive to all NKR-5-3 enterocins) were observed from these incubation temperatures (Fig. 3B). Highest bacteriocin productivities were observed at 25 C despite comparable growth of the producer strain at 30 C. Furthermore, bacteriocin productions were significantly hampered beyond 30 C incubation temperature and bacteriocin productions were not detected at 40 C despite considerable cell growth (Fig. 3).
3 2 1 0 0
2
4
6
8 10 12 14 16 18 20 Incubation time (h)
FIG. 2. Growth and production profiles of multiple bacteriocins of E. faecium NKR-5-3. Bacteriocin productions in nutritionally simple (TSBYE) (A) and nutritionally complex (M17) cultivation media (B). Productions of NKR-5-3 enterocins are shown as follows: closed circles, entNKR-5-3A; closed triangles, entNKR-5-3B; closed squares, entNKR-5-3C; and open circles, entNKR-5-3D. Growth profiles of strain NKR-5-3 in these cultivation media (C); M17 (closed circles) and TSBYE (closed triangles) are shown.
494
PEREZ ET AL.
J. BIOSCI. BIOENG.,
FIG. 3. Influence of incubation temperature on the production of multiple bacteriocins of E. faecium NKR-5-3 in M17 medium after 24-h incubation. It was evaluated using the developed ESI-LC/MS-based quantification system (A) and using conventional antimicrobial assay (spot-on-lawn) (B). Productions of NKR-5-3 enterocins are shown as colored bars; black, entNKR-5-3A; dark gray, entNKR-5-3B; gray, entNKR-5-3C; light gray, entNKR-5-3D. Whereas bacteriocin activity against indicator strains are indicated as colored bars; black, B. subtilis JCM 1465T; and light gray, E. faecalis JCM 5803T. Growth of strain NKR-5-3 is indicated as closed circles.
enterocins. Using the developed method, it was found out that the enterocin NKR-5-3D cannot induce the production of enterocin NKR-5-3B (Fig. 4B). Interestingly, even without exogenous enterocin NKR-5-3D, strain NKR-5-3 in TSBYE medium can synthesize w180 nM (0.5 mg/ml) of enterocin NKR-5-3D but production of other NKR-5-3 enterocins were not induced (Fig. 2A).
DISCUSSION
3200
A
3
2 1600 1
0
0 0 0.5 1 2 3 4 5 10 50 EntNKR-5-3D concentration added (nM)
Cell density (OD600)
Bacteriocin activity (AU/ml)
E. faecium NKR-5-3 produces multiple bacteriocins including a bacteriocin-like production-inducing peptide (8). Some LAB strains have been reported to produce multiple bacteriocins (11,25). This is believed to be an important element in competition among bacteria since these bacteriocins have slightly different mode of actions which is important to the complementation of antimicrobial effects, thus widening the target cell spectrum and/or increasing the effectiveness of the target cell inhibition. It is also believed that multiple bacteriocin production can help the producer strain to abolish resistance problem of some target strains (26). In this paper, the regulation mechanism of multiple bacteriocin production of strain NKR-5-3 was studied by monitoring the production profiles of each enterocin as influenced by different fermentation conditions as well as influenced by its own bacteriocin-like inducing peptide. We developed and optimize a twostep ESI-LC/MS-based quantification system that can effectively monitor the production of each NKR-5-3 enterocin as influenced by various fermentation conditions.
Bacteriocin production (µg/ml)
Sample pre-treatment in LC/MS analysis is essential for the concentration of bacteriocins as well as the removal of mediumborne impurities (22). Moreover, proper separation of the elution of each bacteriocin is equally essential since co-elution would compromise the reliability of the measurements. The presence of co-eluting compounds could lead to under- (ion suppression) or over-estimation (ion enhancement) of the concentrations of the target molecule (27). Analysis of the mass chromatograms showed that impurity peaks were eluted before the elution of the target peptides (data not shown). Furthermore, columns other than copolymer-based columns resulted in co-elution of some NKR-5-3 enterocins, especially enterocins NKR-5-3 A and C (data not shown). Thus selection of the appropriate column for the analysis is very crucial in this developed system. In the case of NKR-5-3 enterocins, copolymer-based columns are effective in separating the elution of each peptide (Fig. 1). The conventional method for monitoring bacteriocin production, antimicrobial assays, can only predict the total bacteriocin productivity rather than individually. This limits the application of the conventional antimicrobial assay in monitoring multiple bacteriocin productions especially if the researcher wants to observe the production profile of the individual bacteriocins. Nevertheless, in cases where the bacteriocins have no overlapping antimicrobial activity, where an indicator microorganism is only sensitive to a single bacteriocin, the conventional antimicrobial assay has the advantage over the developed system. Unfortunately, the NKR-5-3 enterocins have overlapping antibacterial activity (8). The obvious advantages of the developed system over the conventional bacteriocin quantification antimicrobial assay are (i)
8
B
6 4 2 0 0
0.5
1
2
3
4
5
10
50
EntNKR-5-3D concentration added (nM)
FIG. 4. Induction of the production of multiple bacteriocins in TSBYE medium by one of its own peptide, enterocin NKR-5-3D. It was evaluated using conventional antimicrobial assay against E. faecalis JCM 5803T as indicator strain (spot-on-lawn) (A) and using the developed ESI-LC/MS-based quantification system (B). Vertical arrow indicates the threshold concentration (2 nM) of enterocin NKR-5-3D to induce bacteriocin production. Bacteriocin activity against the indicator strain is shown as closed circles while growth of strain NKR5-3 is shown as closed triangles. Whereas bacteriocin productions are indicated as follows: closed circles, entNKR-5-3A; closed triangles, entNKR-5-3B; closed squares, entNKR-53C; and open circles, entNKR-5-3D.
better data quality (visualizability), (ii) higher accuracy, and (iii) pin-points production profiles specific to each peptide. It has been demonstrated previously that fermentation conditions such as cultivation media and incubation temperature influence the bacteriocin production in strain NKR-5-3 (8). In TSBYE medium, only enterocin NKR-5-3B was detected at the onset on logarithmic growth phase (after 4 h) and minute amount of the inducing peptide during the later growth phase (Fig. 2A) while in M17 medium all enterocins were detected after 6 h of growth (Fig. 2B). Interestingly, the growth of strain NKR-5-3 in these media showed that it took longer time for the cells to establish itself in M17 than in TSBYE despite the fact that M17 medium is a more nutritious than TSBYE (Fig. 2C). This phenomenon does make biological sense since in M17 all bacteriocins were synthesized, the utilization of energy was focused on bacteriocin biosynthesis thereby compromising its growth. On the other hand, since in TSBYE only enterocin NKR-5-3B was synthesized at the early stage, more energy was available for growth. The much higher growth in M17 than in TSBYE at the end of fermentation supports this hypothesis (Fig. 2C). The synthesis of bacteriocin requires high amount of energy for both polymerization of building blocks (amino acids) and specific and coordinated control of bacteriocin gene expression (28). The high ATP requirement of bacteriocin synthesis further underlines its primary metabolite nature (29,30). Bacteriocin production in strain NKR-5-3 is dependent with incubation temperature (8). The optimal incubation temperature of NKR-5-3 peptides except enterocin NKR-5-3B, appeared to be at 25 C and productions are significantly reduced beyond 30 C. Enterocin NKR-5-3B appeared to have a wider range of incubation temperature from 20 C to 30 C (Fig. 3A). This phenomenon makes biological sense, since most bacteriocins display low potency at and above 37 C, especially the class IIa bacteriocins (15). A similar phenomenon was observed in a multiple bacteriocin producing strain E. faecium L50, where its bacteriocins have different optimal incubation temperatures. Maximum enterocins L50A and L50B production were detected at 25 C, while enterocins P and Q were maximally produced at 37 C and 47 C respectively (14). Strain NKR-5-3 produces a bacteriocin-like production-inducing peptide referred here as enterocin NKR-5-3D. The induction process activates after a threshold concentration of 2 nM of the inducing peptide is reach (Fig. 4). Similar threshold concentrations of the inducing peptides were reported in some LAB strains. Lactobacillus plantarum C11 requires at least 1 ng/ml (w5 nM) of the inducing peptide PlnA (12), while L. sakei LTH673 requires 0.2 ng/ml (w1 nM) of the inducing peptide IF for the induction mechanism to commence (31). However, in the case of L. sakei 5 which produces an inducing peptide IP-TX in which the inducing peptide of strain NKR-5-3, enterocin NKR-5-3D has 89% homology, requires a higher concentration of 400 ng/ml (w140 nM) to induce the bacteriocin production (11). However, in their study they investigated the induction of bacteriocin production at a high incubation temperature of 37 C, which is beyond the optimal condition for bacteriocin production. Enterocin NKR-5-3D induces the production of the strain NKR5-3 enterocins during the early growth phase and cannot induce its production once the cells already reached the stationary phase. Despite in the high concentration of enterocin NKR-5-3D, constant antibacterial activity was observed when it was added until 6 h (late log phase) of growth but antibacterial activity decreased tremendously when enterocin NKR-5-3D was added at 8 h (early stationary phase) (Fig. 5). This phenomenon makes sense since transcription process of genomic genes including those involved in three-component signal transducting system occurs during the early stage of growth (28). This finding is critical for the study of the regulation mechanism of the production of NKR-5-3 enterocins. In TSBYE medium, strain NKR-5-3 produces approximately 180 nM of
MONITORING OF MULTIPLE BACTERIOCINS BY LC/MS
495
12800
Bacteriocin activity (AU/ml)
VOL. 114, 2012
6400
0 Control
2
4
6
8
Induction time (h) FIG. 5. Growth-phase-dependency of the inducing activity of enterocin NKR-5-3D. Equal specific concentration (2.11 mM/OD600) of enterocin NKR-5-3D was added at different period of growth of E. faecium NKR-5-3 in TSBYE medium. Indicator microorganism used for the assay was E. faecalis JCM 5803T.
enterocin NKR-5-3D, which is more than enough concentration to induce the production of all its peptides yet it did not synthesize all the NKR-5-3 enterocins. Production of enterocin NKR-5-3D in TSBYE was only detected after 10 h of incubation (Fig. 2B). The growth-phase-dependency of the induction process in strain NKR5-3 may offer a logical explanation of this phenomenon. Similar phenomenon was observed by Brurberg et al. (32), their L. sake LTH673 strain loses the inducibility in the late logarithmic phase. They noted that activation of the auto-induction loop depends on the timely accumulation of the inducing peptide. In conclusion, this paper reports the establishment and use of an ESI-LC/MS-based quantification system that can pin-point the production of each NKR-5-3 enterocin as influenced by various fermentation conditions. To the best of our knowledge, this work is the first report on the use of ESI-LC/MS as a quantification tool in monitoring multiple bacteriocin production in lactic acid bacteria. This method proved to be very useful in the understanding the production regulation of the multiple bacteriocin production. ACKNOWLEDGMENTS This work was partially supported by the Ministry of Education, Culture, Sports, Science and Technology (Monbukagakusho) Scholarship Program of Japan, Grant-in-Aid for Scientific Research by the Japan Society for the Promotion of Science (JSPS), the JSPS-National Research Council of Thailand (NRCT) Core University Program on "Development of Thermotolerant Microbial Resources and Their Applications”, and the Research Grant for Young Investigators of Faculty of Agriculture, Kyushu University. References 1. de Vuyst, L. and Leroy, F.: Bacteriocins from lactic acid bacteria: production, purification, and food applications, J. Mol. Microbiol. Biotechnol., 13, 194e199 (2007). 2. Cleveland, J., Montville, T. J., Nes, I. F., and Chikindas, M. L.: Bacteriocins: safe, natural antimicrobials for food preservation, Int. J. Food Microbiol., 71, 1e20 (2001). 3. Riley, M. A. and Wertz, J. E.: Bacteriocins: evolution, ecology, and application, Annu. Rev. Microbiol., 56, 117e137 (2002). 4. Chen, H. and Hoover, D. G.: Bacteriocins and their food applications, Compr. Rev. Food Sci. Food Saf., 2, 82e100 (2003). 5. U.S. Federal Register (U.S. Food and Drug Administration): Nisin preparation: affirmation of GRAS status as a direct human food ingredient. 21 CFR Part 184, Fed. Reg., 53, 11247e11251 (1988). 6. Cotter, P. D., Hill, C., and Ross, R. P.: Bacteriocins: developing innate immunity for food, Nat. Rev. Microbiol., 3, 777e788 (2005).
496
PEREZ ET AL.
7. Wilaipun, P., Zendo, T., Sangjindavong, M., Nitisinprasert, S., Leelawatcharamas, V., Nakayama, J., and Sonomoto, K.: The two-synergistic peptide bacteriocin produced by Enterococcus faecium NKR-5-3 isolated from Thai fermented fish (Pla-ra), Sci. Asia, 30, 115e122 (2004). 8. Ishibashi, N., Himeno, K., Fujita, K., Masuda, Y., Perez, R. H., Zendo, T., Wilaipun, P., Leelawatcharamas, V., Nakayama, J., and Sonomoto K.: Purification and characterization of multiple bacteriocins and an inducing peptide produced by Enterococcus faecium NKR-5-3 from Thai fermented fish. Biosci. Biotechnol. Biochem, 76, 947e953 (2012). 9. Siragusa, G. R. and Cutter, C. N.: Brochocin-C, a new bacteriocin produced by Brochotrix campestris, Appl. Environ. Microbiol., 59, 2326e2328 (1993). 10. Himeno, K., Fujita, K., Zendo, T., Wilaipun, P., Ishibashi, N., Masuda, Y., Yoneyama, F., Leelawatcharamas, V., Nakayama, J., and Sonomoto, K.: Identification of enterocin NKR-5-3C, a novel class IIa bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3. Biosci. Biotechnol. Biochem, 76, 1245e1247 (2012). 11. Vaughan, A., Eijsink, V. G., and van Sinderen, D.: Functional characterization of a composite bacteriocin locus from malt isolate Lactobacillus sakei 5, Appl. Environ. Microbiol., 69, 7194e7203 (2003). 12. Diep, D. B., Håvarstein, L. S., and Nes, I. F.: A bacteriocin-like peptides induces bacteriocin synthesis in Lactobacillus plantarum C11, Mol. Microbiol., 18, 631e639 (1995). 13. Diep, D. B., Axelsson, L., Grefsli, C., and Nes, I. F.: The synthesis of the bacteriocin sakacin A is a temperature-sensitive process regulated by a pheromone peptide through a three-component regulatory system, Microbiology, 146, 2155e2160 (2000). 14. Criado, R., Gutierrez, J., Martin, M., Herranz, C., Hernandez, P. E., and Cintas, L. M.: Immunological characterization of temperature-regulated production of enterocin L50 (EntL50A and EntL50), enterocin P, and enterocin Q by Enterococcus faecium L50, Appl. Environ. Microbiol., 72, 7634e7643 (2006). 15. Fimland, G., Johnsen, L., Axelsson, L., Brurberg, M. B., Nes, I. F., Eijsink, V. G., and Meyer, J. N.: A C-terminal disulfide bridge in pediocin-like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum, J. Bacteriol., 182, 2643e2648 (2000). 16. Leroy, F. and de Vuyst, L.: The presence of salt and a curing agent reduces bacteriocin production by Lactobacillus sakei CTC 494, a potential starter culture for sausage fermentation, Appl. Environ. Microbiol., 65, 5350e5356 (1999). 17. Vera Pingitore, E., Salvucci, E., Sesma, F., and Nader-Macías, M. E.: Different strategies for purification of antimicrobial peptides from lactic acid bacteria (LAB), pp. 557e568, in: Mendez-Vilas, A. (Ed.), Communicating current research and educational topics and trends in applied microbiology. Microbiology book series, vol. 2. Formatex, Spain (2007). 18. Korfmacher, W. A.: Foundation review: principles and applications of LC-MS in new drug discovery, Drug Discov. Today Rev., 10, 1357e1367 (2005).
J. BIOSCI. BIOENG., 19. Xu, R. N., Fan, L., Rieser, M. J., and El-Shourbagy, T. A.: Recent advances in high-throughput quantitative bioanalysis by LC-MS/MS, J. Pharmaceut. Biomed., 44, 342e355 (2007). 20. Rose, N. L., Sporns, P., and McMullen, L. M.: Detection of bacteriocins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Appl. Environ. Microbiol., 65, 2238e2242 (1999). 21. Hindré, T., Didelot, S., Le Pennec, J. P., Haras, D., Dufour, A., and ValleeRehel, K.: Bacteriocin detection from whole bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry, Appl. Environ. Microbiol., 69, 1051e1058 (2003). 22. Zendo, T., Nakayama, J., Fujita, K., and Sonomoto, K.: Bacteriocin detection by liquid chromatography/mass spectrometry for rapid identification, J. Appl. Microbiol., 104, 499e507 (2008). 23. Ghobrial, O. G., Derendorf, H., and Hillman, J. D.: Development and validation of a LC-MS quantification method for the lantibiotic MU1140 in rat plasma, J. Pharm. Biomed. Anal., 49, 970e975 (2009). 24. Fujita, K., Ichimasa, S., Zendo, T., Koga, S., Yoneyama, F., Nakayama, J., and Sonomoto, K.: Structural analysis and characterization of lacticin Q, a novel bacteriocin belonging to a new family of unmodified bacteriocins of grampositive bacteria, Appl. Environ. Microbiol., 9, 2871e2877 (2007). 25. Cintas, L. M., Casaus, P., Herranz, C., Håvarstein, L. S., Hernandez, P. E., and Nes, I. F.: Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q, J. Bacteriol., 182, 6806e6814 (2000). 26. Eijsink, V. G., Axelsson, L., Diep, D. B., Håvarstein, L. S., Holo, H., and Nes, I. F.: Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication, Antonie van Leeuwenhoek, 81, 639e654 (2002). 27. Annesley, T. M.: Ion suppression in mass spectrometry, Clin. Chem. Min. Rev., 49, 1041e1044 (2003). 28. de Vuyst, L., Callewaert, R., and Crabbé, K.: Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavorable growth conditions, Microbiology, 142, 817e827 (1996). 29. Martin, S. A. and Russell, J. B.: Transport and phosphorylation of disaccharides by ruminal bacterium Streptococcus bovis, Appl. Environ. Microbiol., 53, 2388e2393 (1977). 30. Mataragas, M., Drosinos, E. H., Tsakalidou, E., and Metaxopoulos, J.: Influence of nutrients on growth and bacteriocin production by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, Antonie van Leeuwenhoek, 85, 191e198 (2004). 31. Eijsink, V. G., Brurberg, M. B., Middlehoven, P. H., and Nes, I. F.: Induction of bacteriocin production in Lactobacillus sakei by a secreted peptide, J. Bacteriol., 178, 2232e2237 (1996). 32. Brurberg, M. B., Nes, I. F., and Eijsink, G. H.: Pheromone-induced production of antimicrobial peptides in Lactobacillus, Mol. Microbiol., 26, 347e360 (1997).