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Applied and Environmental Microbiology, December 2006, p. 7634-7643, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.00983-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Immunochemical Characterization of Temperature-Regulated Production of Enterocin L50 (EntL50A and EntL50B), Enterocin P, and Enterocin Q by Enterococcus faecium L50{triangledown}

Raquel Criado, Jorge Gutiérrez, María Martín, Carmen Herranz, Pablo E. Hernández, and Luis M. Cintas*

Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain

Received 26 April 2006/ Accepted 9 October 2006


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ABSTRACT
 
Polyclonal antibodies with specificity for enterocin L50A (EntL50A), enterocin L50B (EntL50B), and enterocin Q (EntQ) produced by Enterococcus faecium L50 have been generated by immunization of rabbits with chemically synthesized peptides derived from the C terminus of EntL50A (LR1) and EntL50B (LR2) and from the complete enterocin Q (EntQ) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of these antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect ELISA (CI-ELISA). The NCI-ELISA was valuable for detecting anti-EntL50A-, anti-EntL50B-, and anti-EntQ-specific antibodies in the sera of the LR1-KLH-, LR2-KLH-, and EntQ-KLH-immunized animals, respectively. Moreover, these antibodies and those specific for enterocin P (EntP) obtained in a previous work (J. Gutiérrez, R. Criado, R. Citti, M. Martín, C. Herranz, M. F. Fernández, L. M. Cintas, and P. E. Hernández, J. Agric. Food Chem. 52:2247-2255, 2004) were used in an NCI-ELISA to detect and quantify the production of EntL50A, EntL50B, EntP, and EntQ by the multiple-bacteriocin producer E. faecium L50 grown at different temperatures (16 to 47°C). Our results show that temperature has a strong influence on bacteriocin production by this strain. EntL50A and EntL50B are synthesized at 16 to 32°C, but production becomes negligible when the growth temperature is above 37°C, whereas EntP and EntQ are synthesized at temperatures ranging from 16 to 47°C. Maximum EntL50A and EntL50B production was detected at 25°C, while EntP and EntQ are maximally produced at 37 and 47°C, respectively. The loss of plasmid pCIZ1 (50 kb) and/or pCIZ2 (7.4 kb), encoding EntL50A and EntL50B as well as EntQ, respectively, resulted in a significant increase in production and stability of the chromosomally encoded EntP.


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INTRODUCTION
 
Bacteriocins produced by lactic acid bacteria (LAB) constitute a large and heterogeneous group of ribosomally synthesized proteins or peptides displaying antimicrobial activity against a broad range of gram-positive bacteria, including spoilage and food-borne pathogenic microorganisms (13, 39, 54). LAB bacteriocins may be categorized into three classes: (I) the lantibiotics, or posttranslationally modified peptides; (II) the nonmodified, small, heat-stable peptides; and (III) the large, heat-labile protein bacteriocins. Class II bacteriocins are further grouped into three subclasses: the subclass IIa (pediocin-like bacteriocins containing the N-terminal conserved motif YGNGVxC), the subclass IIb (two-peptide bacteriocins), and the subclass IIc (other peptide bacteriocins) (22, 26, 62). Most bacteriocins are synthesized as biologically inactive precursors containing an N-terminal extension (the so-called double-glycine-type leader sequence or the sec-dependent signal peptide), which is cleaved off concomitantly with secretion of the active bacteriocin (54, 62). During recent years, insight has been gained into the evaluation of the potential use of bacteriocinogenic LAB or their bacteriocins as natural food and feed biopreservatives (14, 34). The bacteriocins produced by enterococci (referred to as enterocins) are particularly active against pathogenic bacteria such as Listeria spp., Clostridium spp., and Staphylococcus spp. (13). Accordingly, bacteriocin-producing enterococci could be exploited in food biopreservation, provided they can be considered safe, mainly due to the antimicrobial activity of the enterocins but also because these microorganisms may play an important role in the ripening and development of aroma and flavor of fermented foods (24, 27).

The potential application of bacteriocins in food biopreservation, either as food additives or produced by starter and/or protective cultures, could be facilitated by optimization of their production and the development of efficient procedures for their detection, quantification, and purification. In general terms, bacteriocin production by LAB is a growth-associated process, occurring throughout the growth phase and ceasing at the end of the exponential phase (41, 57), but the yield of bacteriocin produced may be affected by the producing strain, media composition, and fermentation conditions (58). Moreover, good cell growth does not necessarily result in large bacteriocin production (44, 53). In this respect, biosynthesis of bacteriocins is often stimulated by stress conditions leading to lower growth rates and cell yields but higher bacteriocin activity (19, 55). Considering that laboratory fermentations under optimal conditions differ from real food fermentations, it is of utmost importance to estimate the influence of technological factors and specific environmental conditions that prevail in the food matrix on bacterial cell growth and bacteriocin activity and production (44), as it is of interest to select the optimal growth conditions leading to the maximum bacteriocin activity (1, 32, 42, 44, 58). On the other hand, before approval of the use of bacteriocins in the food industry, analytical methods to determine their presence, activity, and stability in foods should be available (31). In this respect, specific antibodies against bacteriocins can be successfully used for bacteriocin identification and detection by immunochemical assays, such as immunoblotting and enzyme-linked immunosorbent assay (ELISA) (5, 29, 38, 45, 61).

Enterococcus faecium L50, isolated from a Spanish dry fermented sausage (9), produces three class II bacteriocins (four peptides): the subclass IIa sec-dependent enterocin P (EntP); the subclass IIb enterocin L50 (EntL50), consisting of two leaderless antimicrobial peptides with 72% sequence identity, EntL50A and EntL50B; and the subclass IIc leaderless enterocin Q (EntQ). This multiple bacteriocin production confers to E. faecium L50 a broad antimicrobial spectrum against food-borne pathogenic bacteria, such as Listeria monocytogenes, Clostridium perfringens, and Clostridium botulinum, and human and animal clinical pathogens, such as Streptococcus pneumoniae, Streptococcus mitis, Streptococcus oralis, Streptococcus parasanguis, and Streptococcus agalactiae (12). Recently, we have shown that the genetic determinants required for the production of, and immunity to, EntL50, EntQ, and EntP are located on the 50-kb plasmid pCIZ1, the 7.4-kb plasmid pCIZ2, and the chromosome from E. faecium L50, respectively (15). In this work, we describe the generation of specific polyclonal antibodies against chemically synthesized EntQ and the C- and N-terminal fragments of EntL50A and EntL50B as well as EntQ, respectively. Once these antibodies were characterized, specific immunoassays were used to determine the influence of growth temperature (16 to 47°C) on bacteriocin production by the wild-type strain E. faecium L50 and its two bacteriocin-deficient derived mutants, E. faecium L50/30-2 (pCIZ1+, pCIZ2–; EntL50A, EntL50B, and EntP producer) and E. faecium L50/14-2 (pCIZ1–, pCIZ2–; EntP producer) (15).


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MATERIALS AND METHODS
 
Microorganisms, media, and bacteriocin assays.
LAB strains used in this work (Table 1) were grown in MRS broth (Oxoid Ltd., Basingstoke, United Kingdom). Cell-free culture supernatants were obtained by centrifugation of overnight cultures at 12,000 x g at 4°C for 10 min, filter sterilized through 0.2-µm-pore-size filters, and stored at –20°C until use. The antimicrobial activity of cell-free culture supernatants was evaluated by a microtiter plate assay (MPA), performed as previously described by Cintas et al. (12), using as indicator microorganisms Pediococcus acidilactici 347 (EntL50 sensitive, EntP resistant, and EntQ resistant [EntL50s, EntPr, EntQr]) (10), E. faecium T136 (EntL50 sensitive, EntP sensitive, and EntQ resistant [EntL50s, EntPs, EntQr]) (8), and E. faecium L50/30-2 (EntL50 resistant, EntP resistant, and EntQ sensitive [EntL50r, EntPr, EntQs]) (15). One bacteriocin unit (BU) was defined as the reciprocal of the highest dilution of the cell-free culture supernatant causing 50% growth inhibition (50% of the turbidity of the control culture without bacteriocin).


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  		TABLE 1. LAB strains used in this study

Design and synthesis of the antigenic peptides.
The immunogenic potential of EntL50A, EntL50B, and EntQ was evaluated according to their hydrophilicity and antigenic index using a sequence analysis software package (18). The synthetic peptides LR1 (derived from the C terminus of EntL50A), LR2 (derived from the C terminus of EntL50B), Q1 (derived from the N terminus of EntQ), and the chemically synthesized EntQ were selected as antigens for the generation of antibodies. The amino acid sequences of the synthetic peptides were NH2-CAINKIIEWIKKHI-COOH (peptide LR1) and NH2-CTIDQIEKWLKRH-COOH (peptide LR2), where residues in boldface were added to the specific amino acid sequence, and NH2-MNFLKNGIA-COOH (peptide Q1). All peptides were synthesized by 9-fluorenylmethoxycarbonyl chemistry with an Applied Biosystems 413A automated solid-phase peptide synthesizer in the Protein Chemistry Facility at the Centro de Biología Molecular Severo Ochoa (Madrid, Spain). Purity of the peptides was monitored by high-performance liquid chromatography, defined as being higher than 95%, and peptide identity was confirmed by mass spectrometry. Pure EntQ was chemically synthesized at the Molecular Biology Unit (University of Newcastle Upon Tyne, United Kingdom) with a peptide purity of >95% by high-performance liquid chromatography.

Preparation of immunoconjugates and immunization.
Peptides LR1 and LR2 and pure EntQ were conjugated to the carrier protein keyhole limpet hemocyanin (KLH) (LR1-KLH, LR2-KLH, and EntQ-KLH, respectively; 1:2 [wt/wt]) by EDC (1-ethyl-3-[3-dimethylamino-propyl] carbodiimide hydrochloride) coupling using the Imject Immunogen EDC kit (Perbio Science UK Ltd., Cheshire, United Kingdom) for use as immunogens. Similarly, peptide Q1 was conjugated to maleimide-activated KLH (Q1-KLH; 1:2 [wt/wt]) using the Imject Maleimide activated KLH kit (Perbio Science UK Ltd.). The peptides LR1, LR2, and Q1 were also conjugated to ovalbumin (OA) (grade III and fraction VII) (Sigma Chemical Company, St. Louis, MO) in a peptide-OA ratio of 11:1 (mol/mol) by the glutaraldehyde method (4) for use as the solid-phase antigens. Rabbits (New Zealand White females, purchased from a local supplier in Navarra, Spain) were immunized with six doses of the immunogen LR1-KLH (343 µg), LR2-KLH (407 µg), Q1-KLH (392 µg), or EntQ-KLH (362 µg) and emulsified with an equal volume of Freund's adjuvant (Sigma Chemical Company), as previously described (47). Serum was obtained after overnight incubation of blood at 4°C and centrifugation at 1,000 x g at room temperature for 15 min and stored at –20°C until use.

ELISAs.
The ELISA procedures for antisera titration and determination of antiserum specificity and sensitivity were performed basically as previously described (29, 48). Briefly, for antisera titration, flat-bottom 96-well polystyrene microtiter plates (Maxisorp; Nunc, Roskilde, Denmark) were coated overnight (4°C) with 100 µl of LR1-OA, LR2-OA, Q1-OA, or EntQ-OA (5 µg/ml) in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6) (coating buffer [CB]). After this and each subsequent step, the coated plates were washed nine times with 0.05% Tween 20 in 0.01 M phosphate-buffered saline (PBS; pH 7.4). To reduce nonspecific binding, the wells were blocked with 300 µl of 1% (wt/vol) OA (grade III) in PBS (OA-PBS) at 37°C for 1 h. Next, 50 µl of serially diluted serum was added to each well and incubated at 37°C for 1 h. Goat anti-rabbit immunoglobulin G (IgG) peroxidase-labeled conjugate (Cappel Laboratories, West Chester, PA) was diluted 1:500 in OA-PBS, and 100 µl was added to each well. The plates were incubated at 37°C for 30 min. Next, 100 µl of 2,2'-azino-bis[3-ethylbenzthialzoline-6-sulfonic acid] (ABTS) (Sigma Chemical Company) solution with 0.066% (vol/vol) H2O2 was added and the plates were incubated at room temperature in the dark, after which the reaction was stopped by addition of 45.7 mM citric acid plus 0.1% (wt/vol) natrium azide. The amount of bound peroxidase was determined by measuring the absorbance at 405 nm in a Labsystems iEMS reader (Helsinki, Finland) with a built-in software package for data analysis. The titer of each serum sample was arbitrarily set as the maximum dilution that yielded at least twice the absorbance of the same dilution of nonimmune control serum.

For determination of the specificity and sensitivity of the generated antisera, noncompetitive indirect enzyme-linked immunosorbent assays (NCI-ELISAs) were designed. In these assays, wells of the microtiter plates were coated with 100 µl of different concentrations of LR1-OA, LR2-OA, Q1-OA, and EntL50 (EntL50A and EntL50B) purified from E. faecium L50 as previously described (11), as well as in vitro-synthesized EntL50A and EntL50B (11), pure chemically synthesized EntQ, OA, and filter-sterilized supernatants from EntL50, EntP, EntQ, enterocin A, enterocin B, enterocin AS48, sakacin A, sakacin P, pediocin PA-1, and nisin A producer strains in CB. The plates were incubated at 4°C overnight and then were blocked and washed as described for the antiserum titration procedure. Next, 50 µl of anti-LR1-KLH, anti-LR2-KLH, anti-Q1-KLH, or anti-EntQ-KLH antiserum (diluted 1:500, 1:500, 1:200, and 1:500, respectively, in PBS) was added, and the plates were incubated at 37°C for 1 h. After a washing step and the addition of goat anti-rabbit IgG peroxidase conjugate (diluted 1:500 in OA-PBS), the amount of bound peroxidase was determined with the ABTS substrate as described above. Additionally, a competitive indirect ELISA (CI-ELISA) was designed for the determination of the specificity and sensitivity of the antisera generated against peptide Q1 and EntQ. In this assay, microtiter plates were coated with 100 µl of either Q1-OA or EntQ in CB (0.5 µg/ml) and then blocked and washed as described for the antiserum titration procedure. Next, 50 µl of different concentrations of standards (Q1 or EntQ dissolved in PBS or MRS broth) were simultaneously incubated with 50 µl of the correspondent antiserum (diluted 1:100 in PBS) at 37°C for 1 h. After the washing step and addition of the goat anti-rabbit IgG peroxidase, the bound peroxidase was determined with the ABTS substrate as described above. Relative antibody affinity was arbitrarily set as the bacteriocin concentration required to inhibit antibody binding by 50%. For all immunoassays cited above, the concentrations of antibodies, hapten conjugates, or enzyme tracers were optimized by checkerboard titration. Competition curves were obtained by plotting absorbance against the logarithm of the analyte concentration. Sigmoid curves were fitted to a four-parameter logistic equation by using the Labsystems software package (Genesis version 1.60).

Detection and quantification of EntL50A, EntL50B, EntP, and EntQ production by microbiological and immunochemical assays.
The wild-type strain E. faecium L50 was grown in MRS broth (500 ml) at 16, 25, 32, 37, 42, and 47°C under aerobic conditions for 72 h. Similarly, the bacteriocin-deficient mutants E. faecium L50/30-2 and E. faecium L50/14-2 were grown at 25, 37, and 47°C to compare enterocin production with that of the wild-type strain at temperatures at which, according to our previous data (12), (i) maximum enterocin production is achieved and (ii) production of some enterocins is switched off. Bacterial growth (A600), pH, bacteriocin activity, and enterocin concentrations were determined periodically by duplicate. For determination of bacteriocin activity, the cell-free supernatants were assayed by the MPA described above. For enterocin quantification, selected NCI-ELISA conditions were applied to plates coated with known concentrations of purified enterocins and twofold dilutions of the supernatant of an overnight culture of E. faecium L50 grown in MRS broth at 32°C (designed as the control supernatant) to plot the standard curves of enterocin concentration in the supernatant as a function of the absorbance levels at 405 nm. The presence of EntL50A, EntL50B, EntP, and EntQ in the cell-free culture supernatants was then detected and quantified using the bacteriocin-specific antibodies generated in this work and those specific for EntP obtained in a previous work (29) and with the NCI-ELISA, essentially performed as described above. All plates included control wells coated with (i) CB:MRS broth to set the background level of the plate and (ii) six twofold dilutions of the control supernatant to set a standard curve within each plate. Enterocin concentrations were determined by duplicate.


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RESULTS
 
Sensitivity of rabbit anti-peptide antibodies for EntL50A, EntL50B, and EntQ.
The C- or the N-terminal regions within the known amino acid sequence of EntL50 (EntL50A and EntL50B) and EntQ, respectively, were selected for production of chemically synthesized peptides. In addition, pure EntQ was chemically synthesized. Synthetic peptides LR1 (EntL50A, amino acid residues 32 to 44), LR2 (EntL50B, amino acid residues 32 to 43), pure EntQ conjugated to KLH by EDC coupling, and the synthetic peptide Q1 (EntQ, amino acid residues 1 to 9) conjugated to maleimide-activated KLH were used to immunize rabbits. After the final bleeding, the apparent titer in serum of animals immunized with EntQ-KLH and LR2-KLH was 1/25,600, that of those with Q1-KLH ranged from 1/3,200 to 1/6,400, and that of those with LR1-KLH ranged from 1/51,200 to 1/102,400. The sensitivity of the anti-Q1-KLH, anti-EntQ-KLH, anti-LR1-KLH, and anti-LR2-KLH antibodies for EntQ, EntL50A, and EntL50B, respectively, was determined by NCI-ELISA or CI-ELISA. Figure 1 shows the results obtained with the most sensitive sera for detection of pure bacteriocins in either CB or CB:MRS broth. The anti-Q1-KLH sera were not able to detect EntQ by the NCI-ELISA (results not shown), whereas the anti-EntQ-KLH sera recognized this bacteriocin with a detection limit of 15 ng/ml in both CB and CB:MRS broth. By using a CI-ELISA, it was shown that the anti-Q1-KLH serum failed to recognize EntQ, whereas the anti-EntQ-KLH serum recognized EntQ, with an average detection limit of 5 ng/ml, but not the peptide Q1. Remarkably, recognition of EntQ in the CI-ELISA was higher when competition was in PBS:MRS broth than in PBS, as deduced from the smaller amount of free EntQ required for 50% binding inhibition when antigens were dissolved in PBS:MRS broth. By using an NCI-ELISA, the detection limits for EntL50A and EntL50B with the anti-LR1-KLH and anti-LR2-KLH sera, respectively, were 2.5 ng/ml in CB and 1 ng/ml in CB:MRS broth.


Figure 1
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  		FIG. 1. NCI-ELISA detection of purified EntL50 (EntL50A and EntL50B) and EntQ in CB ({diamond}) and CB:MRS broth ({blacklozenge}) using the anti-LR1-KLH (A), anti-LR2-KLH (B), and anti-EntQ-KLH (C) antibodies.

Immunoreactivity of the anti-peptide antibodies to different bacteriocins.
The specificities of the anti-LR1-KLH, anti-LR2-KLH, and anti-EntQ-KLH antibodies in neutralized and filter-sterilized supernatants of 16-h cultures of representative LAB strains, including the bacteriocin-deficient mutants derived from E. faecium L50 and the heterologous EntQ producer Lactobacillus sakei Lb790 (pRCG03) (15), were evaluated by an NCI-ELISA (Table 2). The anti-LR1-KLH and anti-LR2-KLH antibodies showed a high cross-reactivity with supernatants of cultures of the wild-type strain E. faecium L50 and the bacteriocin-deficient derived mutant E. faecium L50/30-2, both EntL50A and EntL50B producers, while the anti-EntQ-KLH antibodies showed a high cross-reactivity uniquely with E. faecium L50 and L. sakei Lb790 (pRCG03). However, all these antibodies showed negligible or no reaction against supernatants of cultures of the bacteriocin-deficient derived mutant E. faecium L50/14-2 (EntP producer) and those of strains producing pediocin PA-1, enterocin A, enterocin B, enterocin P, enterocin AS48, sakacin A, sakacin P, or nisin A. The anti-LR1-KLH and anti-LR2-KLH antibodies showed a high cross-reactivity with in vitro-synthesized EntL50A and EntL50B, respectively. However, the anti-LR1-KLH and anti-LR2-KLH antibodies showed no reaction against in vitro-synthesized EntL50B and EntL50A, respectively. This result is not unexpected, since both antibodies were raised against the amino acid sequences of the more heterogeneous C-terminal fragments of these bacteriocins.


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  		TABLE 2. Reactivities of anti-LR1-KLH, anti-LR2-KLH, and anti-EntQ-KLH serum polyclonal antibodies against culture supernatants of LAB strains as determined by an NCI-ELISA

Temperature-dependent production of enterocins by E. faecium L50.
The influence of temperature in the range of 16 to 47°C on bacterial growth, pH, and bacteriocin production and activity was periodically assessed in supernatants of E. faecium L50 grown in MRS broth. E. faecium L50 grew at 16, 25, 32, 37, 42, and 47°C, reaching the maximum growth (A660 = 1 to 1.2) after 60, 20, 12, 10, 8, and 8 h of incubation, respectively (Fig. 2). The initial pH of the medium (6.2) dropped gradually to 4.2 to 4.8 at the end of the incubation period (results not shown). The production of EntL50A, EntL50B, EntQ, and EntP by E. faecium L50 was parallel to cell growth, starting at the beginning of the exponential phase of growth, and the maximum enterocin concentrations were detected at the late stage of exponential growth or the beginning of the stationary phase (Fig. 3).


Figure 2
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  		FIG. 2. Growth of E. faecium L50 in MRS broth at 16 ({blacklozenge}), 25 ({blacktriangleup}), 32 ({blacksquare}), 37 ({circ}), 42 (x), and 47°C ({square}).


Figure 3
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  		FIG. 3. Concentration of EntL50A (A), EntL50B (B), EntQ (C), and EntP (D) determined by an NCI-ELISA in supernatants of E. faecium L50 grown in MRS broth at 16 ({blacklozenge}), 25 ({blacktriangleup}), 32 ({blacksquare}), 37 ({circ}), 42 (x), and 47°C ({square}).

(i) EntL50A and EntL50B are maximally produced at 25°C.
The influence of temperature on the production of EntL50A and EntL50B by E. faecium L50 as determined by an NCI-ELISA using the anti-LR1-KLH (specific for EntL50A) and anti-LR2-KLH (specific for EntL50B) antibodies is shown in Fig. 3A and B. The maximum production of EntL50A and EntL50B (Table 3) was found at 25°C (201 and 194 ng/ml, respectively), followed by production at 16°C (68 and 66% of that quantified at 25°C, respectively), 32°C (46 and 37% of that quantified at 25°C, respectively), and 37°C (13 and 16% of that quantified at 25°C, respectively). At temperatures above 37°C, production of EntL50A and EntL50B decreased significantly, its maximum production at 42°C being only 2.1 and 2.2% of that found at 25°C, respectively, while at 47°C it represented only 1.1 and 1.2% of that determined at 25°C, respectively. Similarly, Fig. 4 shows the influence of temperature on EntL50 (EntL50A and EntL50B) antimicrobial activities as determined by an MPA using P. acidilactici 347 (EntL50s, EntPr, EntQr) as indicator microorganism. Higher antimicrobial activities attributed to higher production of EntL50A and EntL50B were observed at lower temperatures (Table 4). The maximum antimicrobial activity of EntL50A and EntL50B was found at 25°C (2,247 BU/ml), followed by that at 16°C (44.5% of that at 25°C), 32°C (19% of that at 25°C), and 37°C (13.5% of that at 25°C). Comparison of the MPA and NCI-ELISA results showed that kinetics of EntL50A and EntL50B antimicrobial activity paralleled that of production of these bacteriocins. However, it is interesting to note that at temperatures above 37°C, no antagonistic activity attributable to EntL50 was found in the supernatants, while EntL50A and EntL50B were immunochemically detected (Table 3), revealing a 14-fold higher sensitivity of the immunoassay.


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  		TABLE 3. Maximum enterocin production by E. faecium L50 grown at different temperatures as determined by an NCI-ELISA


Figure 4
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  		FIG. 4. Antimicrobial activity of culture supernatants from E. faecium L50 grown in MRS broth at 16 ({blacklozenge}), 25 ({blacktriangleup}), 32 ({blacksquare}), 37 ({circ}), 42 (x), and 47°C ({square}) against P. acidilactici 347 (EntL50s EntPr EntQr) as determined by an MPA.


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  		TABLE 4. Maximum antimicrobial activity of culture supernatants of E. faecium L50 grown at different temperatures against selected indicator microorganisms as determined by an MPAa

(ii) EntQ is maximally produced at 47°C.
The influence of temperature on the production of EntQ by E. faecium L50 as determined by an NCI-ELISA using the anti-EntQ-KLH antibodies is shown in Fig. 3C. The maximum production of EntQ (Table 3) was found at 47°C (3,721 ng/ml), followed by that at 42°C (89% of that at 47°C), 37°C (55% of that at 47°C), and 32°C (48% of that at 47°C). At temperatures below 32°C, production of EntQ decreased significantly, the maximum EntQ production quantified at 25°C and 16°C being 16 and 4% of that determined at 47°C, respectively. The influence of temperature on EntQ antimicrobial activity was also determined by an MPA using E. faecium L50/30-2 (EntL50r, EntPr, EntQs) as indicator microorganism (Table 4). Higher antimicrobial activities were detected at the temperature range of 32 to 47°C. The maximum EntQ antimicrobial activity was found at 37°C (121 BU/ml), followed by that at 47°C (96% of the observed at 37°C), 32°C (91% of the observed at 37°C), 42°C (79% of the observed at 37°C), 16°C (61% of the observed at 37°C), and 25°C (57% of the observed at 37°C). Strikingly, EntQ production and the antagonistic activity attributable to this bacteriocin were not comparable, since significant differences in production were observed at similar antimicrobial activities in the temperature range of 32 to 47°C.

(iii) EntP is maximally produced at 37°C.
The influence of temperature on production of EntP by E. faecium L50 as determined by an NCI-ELISA using the anti-P2-KLH antibodies (29) is shown in Fig. 3D. The maximum production of EntP (Table 3) was found at 37°C (8,080 ng/ml), followed by that obtained at 25, 32, and 42°C (ca. 75% of that at 37°C), 47°C (60% of that at 37°C), and 16°C (37% of that at 37°C). Interestingly, at the end of the incubation time (60 to 72 h), EntP concentration was higher at lower temperatures. The lack of an indicator microorganism only sensitive to EntP made impracticable the detection of its antimicrobial activity at 37°C and lower temperatures. However, at temperatures above 37°C, production of EntL50A and EntL50B is significantly reduced and no antimicrobial activity against P. acidilactici 347 is detected. This suggests that the antagonistic activity against E. faecium T136 (EntL50s, EntPs, EntQr) could be specifically ascribed to EntP, the maximum antimicrobial activity being higher at 42°C (2,222 BU/ml) than at 47°C (705 BU/ml) (Table 4). Nevertheless, it cannot be ruled out that the small amount of EntL50A and EntL50B detected at 42 to 47°C may still inhibit growth of E. faecium T136 and/or act synergistically with EntP, masking the specific EntP antimicrobial activity.

Differences in bacteriocin production by E. faecium L50 and the bacteriocin-deficient derived mutants E. faecium L50/30-2 and E. faecium L50/14-2.
Growth and production of enterocins by the two bacteriocin-deficient mutants E. faecium L50/30-2 (EntP, EnL50A, and EntL50B producer) and E. faecium L50/14-2 (EntP producer) were also determined at 25, 37, and 47°C in order to compare the results with those obtained with the wild-type strain E. faecium L50 (EntP, EnL50A, EntL50B, and EntQ producer). At these temperatures, these strains grew and produced the bacteriocin(s) encoded by their genomes. However, differences in bacteriocin production by the bacteriocin-deficient mutants and the wild-type strain were found (Table 5). Production of EntL50A and EntL50B by the EntQ-deficient strain E. faecium L50/30-2 and the wild-type strain E. faecium L50 showed only slight differences. However, significant differences in the production of EntP were observed. Maximum EntP production by E. faecium L50/14-2 at 25, 37, and 47°C was 10, 23, and 69% higher than that by E. faecium L50, while maximum EntP production by E. faecium L50/30-2 at 25, 37, and 47°C was 3, 11, and 26% higher than that by the wild-type strain. It is noteworthy that the maximum amount of EntP produced by E. faecium L50/14-2 and E. faecium L50/30-2 at 25°C was maintained during 30 h of further incubation (99.9 and 80.4% of the maximum production, respectively) (results not shown), whereas in the case of E. faecium L50, EntP concentration during this period declined to 37% of the maximum value (Fig. 3).


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  		TABLE 5. Maximum enterocin production by the wild-type strain E. faecium L50 and the bacteriocin-deficient derived mutants E. faecium L50/30-2 and E. faecium L50/14-2 grown at different temperatures as determined by an NCI-ELISA


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DISCUSSION
 
In spite of the multiple advantages of immunochemistry-based methods routinely used as analytical tools in many areas of research, its use in the bacteriocin research field has not been extensively evaluated. Nevertheless, the generation of antibodies against different LAB bacteriocins and the development of appropriate immunoassays have been recently reported for a variety of applications, such as quantification of bacteriocin production by wild-type and recombinant strains, immunopurification, differentiation between active and inactive bacteriocin forms, and bacteriocin immunolocation in food matrix and producer and sensitive bacterial cells (5-7, 16, 29, 38, 49, 50, 59-61). Anti-bacteriocin antibodies have been generated by immunization of animals with a carrier protein conjugated to the whole bacteriocin molecule (16, 61) or to short chemically synthesized fragments deduced from the N- or C-terminal region of the bacteriocins (29, 38, 47-49, 51, 60, 61). It is noteworthy that the success in the generation of specific antibodies against bacteriocins depends on the selection of antigenic fragments, carrier proteins, and conjugation sites and procedures and the subsequent development of appropriate immunoassay formats, which should be carefully performed according to the particularities of each bacteriocin (29, 47, 63). Therefore, in this work, the antigenic peptide fragments LR1, LR2, and Q1 and the pure chemically synthesized EntQ were used, after conjugation to KLH, as immunogens to raise rabbit polyclonal-specific antibodies for EntL50A, EntL50B, and EntQ.

The sensitivity and specificity of the generated antibodies were evaluated by an NCI-ELISA or a CI-ELISA. The anti-LR1-KLH, anti-LR2-KLH, and anti-EntQ-KLH sera were able to detect EntL50A, EntL50B, and EntQ, respectively, by an NCI-ELISA, whereas the anti-Q1-KLH serum did not detect EntQ either by an NCI-ELISA or by a CI-ELISA, probably because the epitope was too short to induce an antibody response (28, 51). The CB:MRS broth enhanced immunochemical detection of EntL50A, EntL50B, and EntQ to a level similar to that described for detection of pediocin PA-1 by a competitive direct ELISA (47) but different from that for the detection of enterocin A (49), pediocin PA-1 (48), and enterocin P (29) by an NCI-ELISA. The pH of the medium (CB or CB:MRS broth) and the possible effect of the Tween 80-containing MRS in preventing the formation of aggregates of the bacteriocin molecules (35, 53, 56) could explain the enhancement of bacteriocin detection in the CB:MRS broth. On the other hand, the CI-ELISA increases the detection of free EntQ, as described for detection of pediocin PA-1 (47, 48) and enterocin B (61), but unlike the results obtained for detection of enterocin A (49) and enterocin P (29). Once again, these results underscore the importance of the development of appropriate immunoassay formats for detection of each specific bacteriocin (47, 63). Of interest is the high specificity (Table 2) and sensitivity of the antibodies generated in the present work, which show a detection limit by an NCI-ELISA of 1 ng/ml (for EntL50A and EntL50B) and 15 ng/ml (for EntQ). These values are similar to the best reported data for nisin A and nisin Z (7, 23) but are considerably lower than most data described for other bacteriocins (16, 29, 38, 45-49, 61).

In this work, the combination of immunochemical and microbiological assays has provided valuable information on the effect of temperature on enterocin production by E. faecium L50. Production of EntL50A, EntL50B, EntP, and EntQ was observed from the beginning of the exponential phase of growth, and the highest enterocin concentrations were detected at the late logarithmic phase or the beginning of the stationary phase (Fig. 2; Table 3), as described for most bacteriocins, such as enterocin 1146 (enterocin A) (57), enterocin 900 (enterocin B) (25), and enterocin 7C5 (64). Temperature has been shown to have a non-growth-related influence on bacteriocin production by some LAB strains (43). Therefore, growth at suboptimal temperatures sometimes leads to an increase in bacteriocin production (41, 58). In this sense, production of EntL50A, EntL50B, EntQ, and EntP by E. faecium L50 seems to be a temperature-dependent process, with temperature having little effect on the final biomass of the producer strain but considerable influence on the volumetric production of the three bacteriocins (four peptides).

Production of EntL50A and EntL50B as well as EntP was maximal at 25 as well as 37°C, respectively, and was reduced with increasing as well as decreasing temperatures, respectively. However, production of EntQ was maximal at 47°C and diminished with decreasing temperatures. Similar to results observed for EntL50A and EntL50B, production of mesenterocin 52A by Leuconostoc mesenteroides subsp. mesenteroides FR52 (40), amylovorin L471 by Lactobacillus amylovorus DCE471 (19, 41), curvacin A by Lactobacillus curvatus LTH1174 (52, 65), sakacin K by L. sakei CTC494 (53), sakacin A by L. sakei Lb706 (20), sakacin P by L. sakei CCUG 42687 (1), lacticin RM by Lactococcus lactis EZ26 (38), and bacteriocin by Lactobacillus pentosus B96 (17) was shown to be stimulated by temperatures unfavorable for growth, particularly low temperatures. Moreover, production of EntL50A and EntL50B is restricted to temperatures below 42°C, although the strain grows at up to 47°C, as observed for mundticin KS production by Enterococcus mundtii NFRI7393 (37). Optimal bacteriocin production at suboptimal temperatures for growth could be ascribed to one or more of the following phenomena: (i) decreased degradation/inactivation of the bacteriocin, (ii) lower rate of enzymatic reactions resulting in reduced growth rate and increased pools of metabolites essential for bacteriocin synthesis, (iii) decreased adsorption of the bacteriocin to the producer cells, (iv) higher gene expression at the transcription and/or the translation level, and (v) higher level of production of the inducing peptide (1, 20, 38, 42, 53). Maximum volumetric EntQ production by E. faecium L50 was observed at 42 and 47°C. To our knowledge, bacteriocin production at similar high temperatures has been previously described only for production of enterocin SE-K4 by Enterococcus faecalis K-4 at 43 to 45°C (21), enterocin GM-1 by E. faecium GM-1 at 35 to 50°C (36), and enterocin by Enterococcus avium PA1 at 45°C (3). Strikingly, the three enterocins (four peptides) synthesized by E. faecium L50 are optimally produced at different temperatures. A similar behavior has been previously described for the multiple-bacteriocin producer strain L. mesenteroides FR52 (40), which produces mesenterocin 52A and mesenterocin 52B maximally at 20 and 25°C, respectively. Considering that temperature may favor production of a desired bacteriocin, it could be feasible to manipulate the ratio of production of different bacteriocins by a multiple-bacteriocin producer by controlling the incubation temperature (58), which may constitute an important factor in the event of a synergistic effect between bacteriocins where an optimal ratio would permit an increase of the total antimicrobial activity (40). After E. faecium L50 entered the stationary phase and the maximum concentration of EntL50A, EntL50B, EntQ, and EntP was achieved, both bacteriocin concentration and antimicrobial activity showed a gradual decrease, which was faster at increasing temperatures. This behavior may be explained by one or more of the following factors: (i) instability of the bacteriocin molecules, (ii) bacteriocin degradation due to protease activity and/or low pH, (iii) aggregation of bacteriocin monomers rendering less active oligomers and/or complexes with other media constituents, and (iv) readsorption of bacteriocins to the producer cell surface at low pH (19, 25, 32, 57, 64).

In the present work, significant differences in the maximum production of EntL50A (0.2 µg/ml), EntL50B (0.2 µg/ml), EntQ (3.7 µg/ml), and EntP (8.1 µg/ml) by E. faecium L50 have been found. The low level of production of EntL50A and EntL50B by this multiple-bacteriocin producer strain is surprising; however, the amounts of EntP and EntQ are quite similar to the values previously reported for production of other LAB bacteriocins such as enterocin A (2.5 µg/ml) by E. faecium T136 (49), pediocin PA-1 (1.9 µg/ml) by P. acidilactici 347 (48), and sakacin P (0.5 to 1 µg/ml) by L. sakei CCUG 42687 (1). Moreover, the maximum production of EntP is similar to that of other EntP producer strains such as E. faecium P13 (7.3 µg/ml), E. faecium AA13 (7.5 µg/ml), and E. faecium G16 (7.9 µg/ml) (29). On the other hand, several reports have shown that identical bacteriocins may be produced in different amounts by different genera, species, and even strains (29, 30, 33, 34, 48, 49, 58, 66). These differences may be explained by (i) the number of copies of the bacteriocin structural genes, (ii) their transcription/translation levels, (iii) the specific activity of maturing enzymes when required, (iv) the level of bacteriocin resistance, and/or (v) the lower level of bacteriocin production by multiple-bacteriocin producer strains (2, 29, 33, 34, 58). In this respect, comparison of enterocin production by the bacteriocin-deficient mutants E. faecium L50/30-2 (pCIZ1+, pCIZ2–; EntP, EntL50A, and EntL50B producer) and E. faecium L50/14-2 (pCIZ1–, pCIZ2–; EntP producer) with that by the wild-type strain E. faecium L50 (pCIZ1+, pCIZ2+; EntP, EntL50A, EntL50B, and EntQ producer) showed significant differences in EntP production (Table 5). Maximum production of EntP was achieved by E. faecium L50/14-2, followed by E. faecium L50/30-2 and E. faecium L50. Strikingly, differences in the maximum EntP production by the wild-type and mutant strains were higher at 37 and 47°C. On the other hand, after the maximum production of EntP by E. faecium L50/14-2 and E. faecium L50/30-2 at 25°C was reached, bacteriocin concentration remained quite stable during further incubation, which differs from the results obtained with E. faecium L50 (Fig. 3). The differences in EntP production described above may be related to the decrease of the metabolic load of E. faecium L50/14-2 and, to a lesser extent, E. faecium L50/30-2 derived from the loss of the pCIZ1 and/or pCIZ2 plasmid burden. In this respect, the lack or reduction of the energy cost associated with plasmid maintenance and replication and with the synthesis of plasmid-encoded bacteriocin(s) may allow these cells, mainly those of E. faecium L50/14-2, to direct more metabolic energy to the production of the chromosomally encoded EntP at higher levels during a longer incubation time. On the other hand, the increased stability of EntP in culture supernatants of strains devoid of pCIZ1 and/or pCIZ2 may be ascribed to the loss of plasmid-encoded proteases and/or proteins interacting with bacteriocin molecules and interfering with their immunochemical detection.

To our knowledge, the present work represents the first report demonstrating the suitability of an immunochemical strategy based on the use of bacteriocin-specific polyclonal antibodies for the characterization of multiple-bacteriocin production by a LAB strain. In this respect, our results show a strong influence of temperature on EntL50 (EntL50A and EntL50B), EntP, and EntQ production by E. faecium L50, although the possibility that the synthesis of each bacteriocin might also be differently regulated by other growth conditions and/or specific environmental signals cannot be ruled out. Therefore, the expanded characterization and optimization of this regulated process, as well as the availability of highly sensitive and specific anti-bacteriocin antibodies, is of scientific and applied interest, since it may facilitate further immunochemically based monitoring and evaluation of the antimicrobial effectiveness of these broad-spectrum bacteriocins in foods to which they are added as biopreservatives.


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ACKNOWLEDGMENTS
 
This work was partially supported by grants 07G/0026/2000 and S-0505/AGR/0265 from the Comunidad de Madrid, Spain, AGL2000-0707 and AGL2003-01508 from the Ministerio de Educación, Cultura y Deporte (MECD), Spain, and PR248/02-11688 from the Fundación Danone/Complutense, Spain. Raquel Criado and María Martín hold fellowships from the MECD. Jorge Gutiérrez was the recipient of a fellowship from the Ministerio de Ciencia y Tecnología (MCYT), Spain.

We thank Ingolf F. Nes (Laboratory of Microbial Gene Technology, Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, Ås, Norway) for providing chemically synthesized EntQ. The help of José M. Martínez and Antonio Basanta is also recognized.


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FOOTNOTES
 
* Corresponding author. Mailing address: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Avda. Puerta de Hierro s/n, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain. Phone: 34-913943751. Fax: 34-913943743. E-mail: lcintas{at}vet.ucm.es. Back

{triangledown} Published ahead of print on 20 October 2006. Back


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Applied and Environmental Microbiology, December 2006, p. 7634-7643, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.00983-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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