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Applied and Environmental Microbiology, April 2004, p. 2098-2104, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2098-2104.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Immunodetection of the Bacteriocin Lacticin RM: Analysis of the Influence of Temperature and Tween 80 on Its Expression and Activity

Tomer Keren, Merav Yarmus, Galia Halevy, and Roni Shapira*

Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Received 1 September 2003/ Accepted 7 January 2004


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ABSTRACT
 
Immunoassays with specific antibodies offer higher sensitivity than do bioassays with indicator strains in the detection and quantification of several bacteriocins. Here we present the purification of lacticin RM and the production of specific polyclonal antibodies to a synthetic peptide resembling an internal fragment of the mature bacteriocin. The specificity and sensitivity of the generated polyclonal antibodies were evaluated in various immunoassays. The detection limits of lacticin RM were found to be 1.9, 0.16, and 0.18 µg ml–1 for Western blot, immuno-dot blot, and noncompetitive indirect enzyme-linked immunosorbent assays, respectively. Immunoassay sensitivities were 12.5-fold higher than that of the agar diffusion test (ADT). The production of lacticin RM showed temperature dependency, with 3, 4.2, 12.7, 28.9, 37.8, and 12 µg ml–1 at 37, 30, 20, 15, 10, and 4°C, respectively. Temperature-stability analysis demonstrated that lacticin RM is sensitive to mild temperature, but the loss of activity does not seem to result from protein degradation. Tween 80 increased the concentration of lacticin RM eightfold and probably affected the results of the ADT either by enhancing the activity of lacticin RM or by increasing the sensitivity of the indicator strain. The use of antibodies for the specific detection and quantification of lacticin RM can expand our knowledge of its production and stability, with important implications for further investigation and future application.


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INTRODUCTION
 
Bacteriocins are antibacterial proteins produced by bacteria that kill or inhibit the growth of closely related bacteria. Many lactic acid bacteria (LAB) produce a high diversity of bacteriocins, with a fairly broad inhibitory spectrum (19). In recent years, many bacteriocins from LAB belonging to different groups have been described, characterized, and purified. Nisin, produced by several strains of Lactococcus lactis, is the most studied bacteriocin, and it is used as a commercial food preservative (6, 11, 16). The role of LAB and their bacteriocins as food biopreservatives is expected to grow in the future as a result of consumer awareness of the potential risks derived from food-borne pathogens as well as from the artificial chemical preservatives currently used to control them (14).

Lacticin RM is a bacteriocin produced by Lactococcus lactis subsp. lactis EZ26 (DSM ID-95-131), isolated from goat's milk. It displays a wide range of inhibition, including Listeria monocytogenes and Staphylococcus aureus (36). The operon encoding lacticin RM is located on a 2.5-kb fragment in the plasmid pHU1, and it is rather simple in comparison to other bacteriocin systems, including only four genes. lacA is the bacteriocin structural gene, while lacF, lacG, and lacI participate in immunity against lacticin RM (36). Preliminary results indicate that lacticin RM is optimally produced at low growth temperatures, with maximal inhibition zones against an indicator strain at 10 and 15°C. Although most of the characterized bacteriocins are produced at the producer's optimal growth temperature, higher production at suboptimal temperatures has been reported for some. However, their molecular regulatory mechanisms have not been thoroughly explored, e.g., those of brevicin 286 (7), amylovorin L471 (10), bavaricin A (22), mesentrocin (20), sakacin P (1, 29), and sakacin A (11).

Bioassays that assess the inhibitory effect of bacteriocins on indicator microorganisms are most commonly used for the detection and quantification of bacteriocin activity. Although the importance of these biologically based methods in the field of bacteriocins is undeniable, they also possess some major drawbacks, such as lack of specificity (35) and low reproducibility (3). The generation of antibodies to bacteriocins has provided specific and sensitive methods for the latter's detection and quantification by enabling the use of immunochemical assays (24, 26-28, 35). Antibodies also offer potential alternative methods for the purification of bacteriocins involving immunoaffinity-chromatography strategies (4, 34).

Previous attempts to purify lacticin RM from milk cultures of L. lactis EZ26 were unsuccessful primarily because unrelated milk proteins were still present (data not shown). Since L. lactis EZ26 shows poor growth ability in more-defined media, we used the host strain Lactococcus lactis subsp. cremoris MG1363, transformed with the plasmid pSY25 (MG1363-pSY25), a lacticin RM producer (36). Here we present the purification of lacticin RM from MG1363-pSY25 cultures.

The production of specific polyclonal antibodies to a synthetic peptide resembling an internal fragment of the mature bacteriocin and the development of immunoassays for the detection and quantification of lacticin RM are described. We also show the use of these methods to study the influence of growth temperature and surfactant addition (Tween 80) on lacticin RM expression and activity.


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MATERIALS AND METHODS
 
Microorganisms, media, and bacteriocin assay.
The plasmid-free strain L. lactis subsp. cremoris MG1363 and MG1363 transformed with the plasmid pSY25 (MG1363-pSY25), a lacticin RM producer (36), were propagated at 30°C in TSY broth (tryptic soy broth supplemented with 0.5% [wt/vol] yeast extract [Difco, Detroit, Mich.] and with 0.05% [vol/vol] Tween 80 [polyoxyethylene sorbitan monooleate; Sigma, Rehovot, Israel]). Pediococcus pentosaceus 43200 (18), a pediocon A producer, was grown at 30°C in MRS (Difco) broth.

To investigate the effect of temperature on lacticin RM production, 50-ml cultures of MG1363-pSY25 were grown at 30°C until mid-log phase (107 CFU ml–1) and then placed in 4, 10, 15, 20, 30, or 37°C temperature-controlled water baths (maximum of 2 min to reach desired temperature) for 94, 48, 28, 10, 7, or 8 h, respectively (until each culture reached the end of the logarithmic phase as judged by optical density at 600 nm). The equal cell number of 109 CFU ml–1 was confirmed by plating serial dilutions of each culture on TSY agar plates. Cell-free supernatant fluids (CSFs) were obtained by centrifugation at 10,000 x g for 5 min at 4°C, filtered through a 0.45-µm-pore-size filter (Millipore, Bedford, Mass.), and stored at –20°C until activity and bacteriocin concentration measurements.

To investigate the effect of Tween 80, a mid-log growing MG1363-pSY25 culture (TSY at 30°C) was divided in two. One half of the culture (10 ml) was supplemented with Tween 80 (0.05% [vol/vol], TSY-T), and the two halves were placed overnight in a 15°C water bath. To the second half of the TSY culture, Tween 80 was added to a final concentration of 0.05% (vol/vol) (to 1.5-ml samples) at different times: 30, 20, 10, and 0 min before CSF collection, and to another aliquot, Tween 80 was added immediately after CSF was collected. All of the cultures were grown to the same cell concentration of 109 CFU ml–1.

Bacteriocin activity was evaluated by the agar diffusion test (ADT) as previously described (4) with some modifications. Buffer A (0.1 M lactic acid buffer [pH 4.5] containing 5% [wt/vol] glycerol) served for the dilution of the CSFs, and 10 µl of each dilution was placed on TSY-2% (wt/vol) agar plates. The overlay consisted of 10 ml of brain heart infusion (Difco) soft agar (1.2%; gives clearer results than does 2%) inoculated with 106 CFU of the indicator strain L. lactis COT21 ml–1 (36). The plates were incubated at 30°C for 16 h or until a clear inhibition zone appeared.

Purification of lacticin RM.
Lacticin RM was purified as previously described for other bacteriocins (5, 24). A 500-ml culture of MG1363-pSY25 was grown at 15°C until the end of the logarithmic phase, and the cells were collected by centrifugation at 10,000 x g for 20 min. The supernatant was filtered in a Corning filter system (Corning Corporation, Corning, N.Y.). Ammonium sulfate precipitation was performed with 36.1 g of ammonium sulfate per 100 ml of filter-sterilized supernatant. The solution was stirred for 1 h at 6°C, and the precipitate was collected by centrifugation at 10,000 rpm for 20 min. The pellet was dissolved in 20 mM citrate buffer, pH 5.2, and desalted with a dialysis membrane with a 3.5-kDa-molecular-mass cutoff against double-distilled H2O (ddH2O), followed by dialysis against 20 mM citrate buffer, pH 5.2.

Cation-exchange chromatography was performed on a 1-ml SP-Sepharose Fast Flow column (Amersham Biosciences, Uppsala, Sweden) equilibrated with 20 mM citrate buffer, pH 5.2. Bacteriocin culture supernatant was diluted with ddH2O to match buffer conductivity. A NaCl stepwise ascending gradient (50, 100, 250, 400, and 500 mM and 1 M, 5 ml each) was applied, and eluents were collected in 1-ml samples. Fractions 1 to 4 of 250 mM NaCl showed bacteriocin activity and were pooled and desalted with a dialysis membrane with a 3.5-kDa-molecular-mass cutoff against ddH2O, followed by dialysis against 20 mM citrate buffer, pH 5.2. The bacteriocin activity during the entire purification process was determined by the ADT method as described above.

SDS-PAGE.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described in the work of Laemmli (21). Proteins were separated in 12.5% acrylamide at 180 V for approximately 45 min, with the Bio-Rad Mini-Protean II electrophoresis apparatus (Bio-Rad Laboratories, Hercules, Calif.). The gel was stained for 1 h with GelCode blue stain reagent (Pierce, Rockford, Ill.) and destained overnight in deionized water. Another gel was assayed for antimicrobial activity essentially as described in the work of Bouksaim et al. (4). Following fixation of the gel for 1 h in a solution of 20% isopropanol-10% acetic acid, the gel was washed for 2 h with deionized water, aseptically placed into a petri dish, and overlaid with brain heart infusion inoculated with L. lactis COT21 as described for the ADT.

Sequence analysis by N-terminal sequencing and mass spectrometry.
Proteins subjected to SDS-PAGE were electroblotted onto a polyvinylidene difluoride pyridine membrane (Immobilon-PSQ; Millipore), with a Bio-Rad Trans-Blot electrotransfer apparatus according to the manufacturer's instructions. The apparent lacticin RM band was excised from the membrane, and N-terminal sequence analysis was performed by Edman degradation on an automated gas-phase sequencer with on-line phenylthiohydantoin derivative identification by reverse-phase high-pressure liquid chromatography at the Weizmann Institute of Science (Rehovot, Israel). In addition, another band was treated with proteases and the resulting peptides were analyzed by ion-trap liquid chromatography-mass spectroscopy. The resulting sequences were compared to that of lacA (36). The final concentration of the purified bacteriocin was estimated with the extinction coefficient of lacticin RM. An A280 of 2.12 corresponded to 1 mg ml–1.

Materials.
The amino acid sequence of an internal fragment of the mature lacticin RM (peptide RM1) used in this work was NH2-NMSQKGIRKTQSSSSMQTYVGTY-COOH. The peptide RM1 (residues 75 to 92; 2,686 Da; 10 mg) was synthesized at MBC, Rehovot, Israel. Goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (IgG-HRP) was obtained from Pierce. Nisin A (2.5%, wt/wt) was purchased from Sigma.

Preparation of immunoconjugates and immunization.
Conjugation of peptide RM1 to maleimide-activated keyhole limpet hemocyanin (KLH) and immunization were performed at MBC. Two rabbits were immunized with RM1-KLH as described previously (28). Rabbits were bled on day 51, and a final bleeding was performed on day 71.

Indirect enzyme-linked immunosorbent assay (ELISA).
For antiserum titration, flat-bottomed polystyrene microtiter plates (Maxisorp; Nalge Nunc International, Rochester, N.Y.) were coated overnight (4°C) with 100 µl of peptide RM1 (1 mg ml–1) in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6) (coating buffer [CB]). The plates were washed four times with 300 µl of washing solution (0.05% Tween 20 in phosphate-buffered saline [PBS; 0.01 M; pH 7]). The plates were blocked for 2 h at 37°C with 300 µl of 5% (wt/vol) skim milk powder (SM) in PBS (SM-PBS) and then washed five times. Next, 100 µl of serially diluted antiserum was added to each well and incubated for 1 h at 37°C. Unbound antibodies were removed by washing four times, and 100 µl of goat anti-rabbit IgG-HRP (diluted 1:5,000 in SM-PBS) was added to each well. The plates were incubated for 30 min at 37°C and washed six times. The amount of bound peroxidase was determined with the tetramethylbenzidine substrate kit (Pierce) as described by the manufacturer. The optical density at 450 nm was determined with an ELX808 reader (Bio-Tek Instruments, Winooski, Vt.). The titer of each serum sample was arbitrarily set as the maximum dilution that yields at least twice the absorbance of the same dilution of nonimmune control serum.

For the determination of antiserum specificity and for quantification of lacticin RM, a noncompetitive indirect ELISA (NCI-ELISA) was employed. Microtiter plates were coated with 100 µl of different concentrations (in CB) of peptide RM1 (0.05 to 0.5 µg ml–1), purified lacticin RM, nisin A (1 to 10 µg ml–1), active CSF of the pediocon A producer (P. pentosaceus 43200), or CSF from MG1363 and MG1363-pSY25. The plates were maintained overnight at 4°C and then blocked and washed as described for the antiserum titration procedure. Next, 100 µl of anti-RM1 antiserum (diluted 1:400 in 1% SM-PBS) was added, and the plates were incubated for 1 h at 37°C. After a washing step and the addition of 100 µl of goat anti-rabbit IgG-HRP (diluted 1:5,000 in 1% SM-PBS), the amount of bound HRP was determined with tetramethylbenzidine substrate as described above. The increase in the absorbance was proportional to the amount of specific antigen in the samples. The lacticin RM concentration in CSFs was calculated using a calibration curve based on purified lacticin RM.

Western blotting.
Proteins subjected to SDS-PAGE were electroblotted onto a nitrocellulose membrane. Nonspecific binding sites were blocked with Tris-buffered saline (TBS; 20 mM Tris base, 0.15 M NaCl [pH 7.6]) plus 5% (wt/vol) SM at room temperature for 2 h on an orbital shaker. The membrane was washed with 0.5% (vol/vol) Tween 20 in TBS (TBS-T) once for 15 min and twice for 10 min. The membrane was incubated first with primary antibody (anti-RM1 antiserum diluted 1:500 with 1% [wt/vol] SM in TBS-T) for 1 h at 37°C. The membrane was washed as described above and then incubated with 20 ml of goat anti-rabbit IgG-HRP (diluted 1:10,000 in the same buffer), for 1 h at 37°C. For visualization, the membrane was treated with West Pico chemiluminescent substrate (Pierce) according to the manufacturer's instructions. Image capture was performed using the ImageMaster VDS-CL system (Amersham Biosciences). The obtained spot intensities were analyzed using NIH Image 1.55 software (National Institutes of Health, Bethesda, Md.). The lacticin RM concentration was calculated for each sample using a calibration curve based on serial dilutions of purified lacticin RM that were separated in a parallel gel.

Immuno-dot blot assay.
From the CSFs, 10 µl was mixed with 90 µl of CB and dripped onto a nitrocellulose membrane, with a Bio-dot SF microfiltration apparatus (Bio-Rad Laboratories). Purified lacticin RM diluted in CB served for the calibration curve. The subsequent blocking, antibody addition, and detection steps were exactly as described above for the Western blot assay. To calculate the concentration of lacticin RM in 1 ml of CSF, the values obtained from the calibration curve were multiplied by 10.


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RESULTS
 
Determination of lacticin RM propeptide sequence.
Lacticin RM was purified from the culture supernatant of L. lactis MG1363 harboring pSY25. Recovery of lacticin RM was 4% of the initial total activity with a 16-fold increase in final specific activity relative to the culture supernatant. The purity of lacticin RM at the final step was analyzed by SDS-PAGE (Fig. 1A), yielding a single band at approximately 11 kDa, with antibacterial activity, as evidenced by the inhibition zone in Fig. 1C. Sequence analysis of the single band was performed, and the sequence was compared to the DNA sequence of lacA (accession number AF080265) (36). The sequence indicated that the mature lacticin RM is 11,064 Da (Fig. 2). Based on the molecular mass and the extinction coefficient, the concentration of lacticin RM at the final purification step was calculated to be 480 µg ml–1.



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  		FIG. 1. SDS-PAGE analysis: protein staining, Western blotting, and antimicrobial activity of lacticin RM. (A) Gel stained with GelCode blue. (B) Western blotting of purified lacticin RM with polyclonal antibodies to peptide RM1. (C) Detection of bacteriocin activity with overlay of L. lactis COT21 in soft agar. Lane 1, molecular mass markers (masses indicated on the left); lanes 3, 5, and 6, purified lacticin RM; lanes 2 and 4, the eluted fraction from culture of a nonproducing strain (MG1363; control), followed by the same purification steps as with the active culture.



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  		FIG. 2. Sequence of preformed lacticin RM. The mature lacticin RM sequence is in boldface, and the cleavage site is marked with an arrow (CDS 325-729 in accession number AF080265). The sequence of fragment RM1, of lacticin RM, used for the immunization, is underlined.

Production of antipeptide antibodies specific to lacticin RM.
A synthetic peptide similar to an internal region of the mature (secreted) lacticin RM sequence was produced, based on antigenic index evaluation with a sequence analysis software package (9). This peptide, RM1, was conjugated to KLH and used to immunize rabbits. At the final bleeding, the animals had an apparent titer in serum of 110,000 to 250,000. The antisera with the highest titers were combined to yield a titer of approximately 140,000 and were used throughout this work.

The specificity of the anti-RM1-KLH antibodies to secreted lacticin RM was first determined by Western blotting (Fig. 1B). The obtained chemiluminescent signal showed specific recognition of the bacteriocin with anti-RM1-KLH antibodies.

To further ascertain the specificity of the antibodies, NCI-ELISA was performed with CSFs of MG1363-pSY25 (lacticin RM producer), MG1363 (nonproducer), P. pentosaceus (pediocon A producer), a solution of nisin A, and a solution of the peptide RM1. Significant absorbance over background was seen only for peptide RM1 and the CSF of MG1363-pSY25 (data not shown). This result indicated that anti-RM1-KLH antibodies are specific for lacticin RM and that there is no cross-reactivity with the other analyzed bacteriocins.

Comparison of various immunoassays for the detection of lacticin RM.
To evaluate the sensitivity of the polyclonal antibodies in the detection of lacticin RM and to verify any advantage over the use of the bioassay, we compared the sensitivities of various immunoassays to the ADT. First, Western blot analysis was performed with serial dilutions of purified lacticin RM (20 µl per well). A detection limit of 1.9 µg ml–1 was found (Fig. 3A). The calculated calibration curve exhibited linear proportions between the band intensities and lacticin RM concentrations in the range of 1.9 to 15 µg ml–1. The detection limit of lacticin RM in the ADT, with L. lactis COT21 as the indicator strain, was quite similar. We were able to detect a visible inhibition zone at roughly 2 µg ml–1 (data not shown), demonstrating approximately the same sensitivity for the Western blot analysis and the bioassay. In contrast to the Western blot assay, the immuno-dot blot assay is not limited as to the amount that can be loaded, and 100 µl per well was applied. A minimal detection level of 0.16 µg ml–1 was found, much lower than that for the Western blot assay (Fig. 3B). Finally, an NCI-ELISA was developed to determine the sensitivity of the anti-RM1 antibodies to lacticin RM. A 100-µl aliquot of twofold dilutions in CB of purified lacticin RM was used in a microtiter plate. The resultant calibration curve indicated that the anti-RM1 antibodies recognize a low concentration of lacticin RM in the NCI-ELISA, similar to that in the immuno-dot blot assay. In the NCI-ELISA, the lower limit was 0.18 µg ml–1 with a linear correlation between 0.18 and 6 µg ml–1 (data not shown).



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  		FIG. 3. Quantitation of purified lacticin RM. Twofold dilutions of purified lacticin RM were subjected to Western blotting (A) and immuno-dot blot detection (B) as described in Materials and Methods.

Lacticin RM is maximally produced at low temperatures.
The optimal temperature for lacticin RM production was determined using the ADT. Higher activity was observed at low temperatures, with maximal activity at 10°C (Table 1 and Fig. 4A). No activity was detected at 37°C, and only a faint clear zone was obtained at 30°C, which is the optimal growth temperature of the host strain MG1363 (13). To determine the actual concentration of lacticin RM produced, the same CSFs as used in the ADT were subjected to immuno-dot blot assay. The average results of three different cultures grown at each temperature are shown in Table 1. An image generated in a representative analysis is shown in Fig. 4B. A negative control, CSF of MG1363 cultured at 15°C, did not show any signal relative to the background (data not shown). NCI-ELISA of the same supernatants displayed approximately the same results (data not shown). Interestingly, immunodetection showed the presence of lacticin RM at 37°C. Based on the ADT, this temperature was thought to be unsuitable for lacticin RM production. This result is an outcome of the higher sensitivity of the immunodetection than of the bioassay.


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  		TABLE 1. Bacteriocin activity and concentration in CSFs produced at different temperaturesa



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  		FIG. 4. Production of lacticin RM at different temperatures. Cultures of MG1363-pSY25 were grown at 30°C to mid-log growth phase and then placed at 37, 30, 20, 15, 10, and 4°C (in temperature-controlled water baths) until each culture reached the end of the logarithmic growth phase. (Each culture contained an equal cell number, 109 CFU ml–1.) CSFs were collected from these cultures, and 10 µl was taken for ADT (A) and immuno-dot blot assay (B).

To determine the effect of temperature on bacteriocin stability, we performed the following test. Aliquots (0.5 ml) of purified lacticin RM (20 µg ml–1) were incubated at 10, 20, 30, 40, and 50°C for 5, 10, 20, and 30 min (in temperature-controlled water baths, maximum of 0.5 min to reach desired temperature). A sample kept at 4°C served as a control. The activity of these samples was determined by ADT (Fig. 5A). The activity of lacticin RM was lost after just 5 min of exposure to 50°C, and there was even a decrease in activity at 10°C after that short time. Extending the incubation for longer times (up to 10 h) did not result in significant reduction beyond the observed decrease after 30 min of incubation. Western blot analysis of samples incubated for 30 min at the different temperatures showed that they displayed equal amounts of protein, which migrated in the gel to the same positions (Fig. 5B). These results demonstrated that lacticin RM is temperature sensitive, even at 10°C, but that loss of activity does not result from protein degradation. This could explain the finding of very low activity in CSF from cultures grown at 30°C and no activity at all in CSF from cultures grown at 37°C, despite expression of the bacteriocin in these cultures, as seen above. Nevertheless, the expression of lacticin RM is much higher at low temperatures given that the low stability at 30 and 37°C could not explain the marked difference found by the immunodetection of lacticin RM in that range of temperatures.



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  		FIG. 5. Temperature stability of lacticin RM. Aliquots of purified lacticin RM (20 µg ml–1) were incubated at 10, 20, 30, 40, and 50°C for 0, 5, 10, 20, and 30 min. A sample kept at 4°C served as a control. Shown are results from ADT (A) and Western blot analysis (B) of the samples that were incubated at the different temperatures for 30 min. AU, activity units.

Influence of Tween 80 on the production of lacticin RM.
Previous reports have shown an increased yield of bacteriocin production when Tween 80 or Tween 20 was added to the growth medium (2, 12, 15). When 0.05% Tween 80 was added to the growth medium of MG1363-pSY25, there was a 5- to 10-fold increase in the inhibition zones, as determined by ADT (data not shown). To understand the role of Tween 80 in the resultant increased activity, we performed the following experiment. Half of a mid-log growing culture of MG1363-pSY25 was supplemented with Tween 80 to a final concentration of 0.05% and was grown overnight at 15°C. The second half was kept overnight under the same conditions (without Tween 80). To four aliquots of the latter half, 0.05% Tween 80 was added at different time points: 30, 20, 10, and 0 min before CSF collection. CSF was also collected from the same overnight culture at the same time point, with no addition of Tween 80. To half of that CSF, Tween 80 was then added (CSF + T). Bacteriocin activity and lacticin RM concentration were determined for the collected samples by the ADT and NCI-ELISA, respectively. In all of the samples that were supplemented with Tween 80 before CSF collection, the activities were similar among samples and sixfold higher than that of CSF from the culture that was not supplemented with Tween 80 (Fig. 6). The initial concentration of lacticin RM in the CSF before Tween 80 supplementation was 6.16 µg ml–1; it increased to approximately 30 µg ml–1 after supplementation, regardless of the time of Tween 80 addition. Upon washing of the cells with 0.01 M PBS plus Tween 80, no significant residual adsorbed bacteriocin was evident (data not shown). This indicated that Tween 80 increases the bacteriocin level by deadsorption of preformed bacteriocin and not by increasing its production or secretion, since the effect of the detergent was instantaneous. We determined the minimal Tween 80 concentration required for maximal deadsorption of lacticin RM to be 0.025% (data not shown).



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  		FIG. 6. Influence of Tween 80 supplementation on bacteriocin concentration (open bars) and activity (solid bars). Tween 80 was added to a final concentration of 0.05% at the indicated times before CSF collection (ON, overnight; CSF + T, immediately after CSF collection; CSF, without Tween 80). Lacticin RM concentrations (micrograms milliliter–1) were determined using NCI-ELISA, and activity (activity units [AU] milliliter–1) was determined using ADT. All of the cultures reached the same cell concentration, 109 CFU ml–1. Values are the means of triplicates. The error bars indicate the standard deviations.

Comparison of the activities of CSF with (CSF + T) and without Tween 80 (Fig. 6) indicated that Tween 80 also enhances the activity of lacticin RM or increases the sensitivity of the indicator strain (equal bacteriocin concentrations in the two samples). When Tween 80 was added to CSF to a final concentration of only 0.01%, the activity increased from 200 to 500 activity units ml–1 (data not shown). Increasing the Tween 80 concentration above that level did not show any enhancement in bacteriocin activity.


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DISCUSSION
 
Lacticin RM was purified and its propeptide sequence was determined in order to select the synthetic peptide sequence for immunization. Compared to those with the purification of other bacteriocins, such as pediocin L50, propionicin PLG-1, pediocin PA-1, and nisin (5, 24, 28, 33), the yield of purified lacticin RM was rather low. Attempts to introduce reverse-phase and hydrophobic-interaction chromatography resulted in loss of activity and were therefore not used in the purification scheme. Nevertheless, SDS-PAGE displayed a single band with bacteriocin activity against the indicator strain L. lactis COT21. Sequence analysis confirmed the N-terminal sequence of the mature lacticin RM. The low purification yield was a major drawback for the immunization of rabbits with the bacteriocin by itself or conjugated to a carrier protein. Based on previous success in generating polyclonal antibodies to chemically synthesized fragments of pediocin PA-1 and enterocin A conjugated to a carrier protein (26-28), we attempted the same approach. Since lacticin RM does not share any similar sequence with other bacteriocins (36), antibodies generated to lacticin RM are predicted to be specific. Indeed, the polyclonal antibodies generated showed specificity only to the RM1 peptide and lacticin RM. There were no signals over background detected for CSFs of MG1363 (nonproducer), P. pentosaceus (pediocin A producer), or a solution of nisin A.

Great differences in the sensitivities of the various immunoassay methods were obtained, with a Western blot assay showing the least sensitivity compared to immuno-dot blot assay and NCI-ELISA. The lower sensitivity obtained by Western blotting can be explained mainly by the likely loss of transferred protein during the electroblotting or by the inaccessible structural conformation of the bacteriocin after transfer to the nitrocellulose membrane. Compared to the detection limits of the ADT, the immuno-dot blot and the NCI-ELISA methods showed 12.5-fold-higher sensitivity. Immunodetection of other bacteriocins has displayed various sensitivity levels, with some presenting much higher sensitivity than others. Bouksaim et al. (4) reported a detection limit of nisin Z with ELISA of 0.75 ng ml–1 in buffer and 3.5 ng ml–1 in milk. Antibodies produced to detect pediocin PA-1 showed a detection limit of 0.01 µg ml–1 with ELISA but only 5 µg ml–1 with immuno-slot blot assay (27). Immunodetection of enterocin A and pediocin PA-1 was able to detect the low concentration of 0.09 µg ml–1 (26). Although our ELISA method showed 2- to 20-fold-lower sensitivity than other methods, it proved sufficient for the detection of lacticin RM in culture medium at concentrations much below the detection limit of the bioassay.

The production of lacticin RM was found to be temperature dependent. The ADT clearly demonstrated higher production at low temperatures, mainly 10 and 15°C. It is important to mention that the wild-type producer L. lactis EZ26 displayed similar bacteriocin production at the analyzed temperatures when grown in milk (data not shown). With the immuno-dot blot assay we could quantitate the concentration of lacticin RM in the CSF, and we detected lacticin RM even at 37°C. The concentrations at 30 and 37°C were above the minimal concentration for inhibition of the sensitive bacteria, as determined with purified lacticin RM. These results illustrate the presence of inactive bacteriocin molecules at these temperatures.

Optimal growth temperature usually results in optimal bacteriocin production (31). However, there are some reports of bacteriocins with maximal production at suboptimal growth temperatures. In the case of amylovorin L471, slow growth at low temperature was suggested to free up more energy for bacteriocin production (10). For sakacin P, higher bacteriocin concentrations at low temperatures were claimed to be due to different rate-limiting reactions dependent upon temperature, resulting in better utilization of carbon and/or energy at low growth rates and increased availability of essential metabolites for bacteriocin synthesis (1). Another explanation was suggested to be increased degradation or inactivation of the bacteriocin at high temperatures (23, 29). An interesting case is the temperature sensitivity of sakacin A, which is regulated by a three-component regulatory system (11), although the optimal temperature presented in that case was not as low as that for sakacin P (29). The instability of production of sakacin A at elevated temperatures was suggested to be the result of reduced synthesis of the pheromone peptide Sap-Ph (11). Here we conclude that higher-temperature inactivation of lacticin RM occurs but does not result in evident degradation, since the size of the bacteriocin in SDS-PAGE is intact. We observed that the decrease in activity takes place in the first 30 min of incubation and that later there is no significant reduction (data not shown). Moreover, it is likely that, when the bacteriocin adsorbs to the agar in the ADT, the bacteriocin confers less temperature sensitivity. Immunodetection of the same concentration of lacticin RM, even after incubation at 50°C, indicated that lacticin RM, whether active or not, displays the actual expressed levels. This implies that lacticin RM production is higher at low temperatures, most likely as a result of higher expression, probably at the level of transcription, translation, or both.

An increase in bacteriocin production as a result of adding from 0.75 to 1% Tween 20 or Tween 80 to the medium has been reported by Garver and Muriana (12) and Huot et al. (15). Nonionic detergents such as Tween 80 may mimic the effect of various food constituents in inducing the production of bacteriocins, and they are known to stimulate protein secretion by affecting membrane fluidity (32). Tween-treated cultures also increased the supernatant activity relative to total activity, probably by desorption and disaggregation of the bacteriocin (2, 17, 30). In contrast to other reports (15, 29), our results indicated that Tween 80 does not stimulate growth or bacteriocin production. We found that Tween 80 reduces the adsorption of lacticin RM to producer cells and that this effect is instantaneous, leaving no significant residual bacteriocin adsorbed to the cells. This effect could be important for increasing the yield of bacteriocin in harvest and purification. Moreover, Tween 80 also increased the sensitivity of the indicator strain to lacticin RM or increased bacteriocin activity. It was shown previously that pediocin AcH possesses higher listericidal activity in slurries of nonfat milk, butterfat, or meat when acting in the presence of Tween 80 (8). In a report by Li et al. (2002), Tween 20 was shown to increase the sensitivity of L. monocytogenes to nisin by increasing the binding of nisin to the cells. Further analysis is needed to conclude that Tween 80 increases sensitivity to lacticin RM and that the mechanism is the same. Regardless of the mode of action, the synergistic effect of Tween 80 or other surfactants with lacticin RM could be useful in various applications.

In this paper, we demonstrate various immunochemical methods and describe their sensitivity and specificity in the detection of lacticin RM. The advantage of the immunomethods over the bioassay in this endeavor is clear, although one should not disregard the complementary information provided by the bioassay, as illustrated in the case of temperature stability and of the effect of Tween 80 on lacticin RM activity. The combined information from the detection methods indicates that lacticin RM expression is temperature dependent. The use of a lacticin RM-producing strain as a protective culture in dairy products could be very attractive. Lacticin RM production would not take place during fermentation but would start with storage, when low temperature is applied. Further study of lacticin RM is important to improve its temperature-controlled expression. The use of the generated antibodies will be central for the investigation of the regulation of lacticin expression. In addition, the antibodies could be utilized for monitoring lacticin RM in complex media and foods.


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ACKNOWLEDGMENTS
 
This work was partially supported by the Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, Rehovot, Israel.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel. Phone: 972-8-9489818. Fax: 972-8-9476189. E-mail: shapira{at}agri.huji.ac.il. Back


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Applied and Environmental Microbiology, April 2004, p. 2098-2104, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2098-2104.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Criado, R., Gutierrez, J., Martin, M., Herranz, C., Hernandez, P. E., Cintas, L. M. (2006). Immunochemical Characterization of Temperature-Regulated Production of Enterocin L50 (EntL50A and EntL50B), Enterocin P, and Enterocin Q by Enterococcus faecium L50. Appl. Environ. Microbiol. 72: 7634-7643 [Abstract] [Full Text]  

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