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Applied and Environmental Microbiology, April 2008, p. 2471-2479, Vol. 74, No. 8
0099-2240/08/$08.00+0     doi:10.1128/AEM.02559-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cloning and Heterologous Production of Hiracin JM79, a Sec-Dependent Bacteriocin Produced by Enterococcus hirae DCH5, in Lactic Acid Bacteria and Pichia pastoris

Jorge Sánchez, Juan Borrero, Beatriz Gómez-Sala, Antonio Basanta, Carmen Herranz, Luis M. Cintas, and Pablo E. Hernández^*

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

Received 13 November 2007/ Accepted 20 February 2008

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Hiracin JM79 (HirJM79), a Sec-dependent bacteriocin produced by Enterococcus hirae DCH5, was cloned and produced in Lactococcus lactis, Lactobacillus sakei, Enterococcus faecium, Enterococcus faecalis, and Pichia pastoris. For heterologous production of HirJM79 in lactic acid bacteria (LAB), the HirJM79 structural gene (hirJM79), with or without the HirJM79 immunity gene (hiriJM79), was cloned into the plasmid pMG36c under the control of the constitutive promoter P₃₂ and into the plasmid pNZ8048 under the control of the inducible P_NisA promoter. For the production of HirJM79 in P. pastoris, the gene encoding the mature HirJM79 protein was cloned into the pPICZA expression vector. The recombinant plasmids permitted the production of biologically active HirJM79 in the supernatants of L. lactis IL1403, L. lactis NZ9000, L. sakei Lb790, E. faecalis JH2-2, and P. pastoris X-33, the coproduction of HirJM79 and nisin A in L. lactis DPC5598, and the coproduction of HirJM79 and enterocin P in E. faecium L50/14-2. All recombinant LAB produced larger quantities of HirJM79 than E. hirae DCH5, although the antimicrobial activities of most transformants were lower than that predicted from their production of HirJM79. The synthesis, processing, and secretion of HirJM79 proceed efficiently in recombinant LAB strains and P. pastoris.

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Bacteriocins are ribosomally synthesized peptides or proteins with antimicrobial activity, and considerable research interest has focused on the bacteriocins produced by lactic acid bacteria (LAB) because of their potential applications in food, pharmaceuticals, nutraceuticals, and veterinary and human medicine (10, 19, 24, 50). Among the LAB, the enterococci produce a diverse and heterogenous group of bacteriocins, coined enterocins, which differ with respect to their antimicrobial activities, structures, processing, and secretion mechanisms (8, 16, 40). However, since enterococci are involved in food spoilage, nosocomial infections, and the spread of antibiotic resistance (36, 41), interest in the heterologous production and functional expression of enterocins in other microbial hosts is growing rapidly. Moreover, the production of enterocins in other hosts could lead to increased bacteriocin production, production of bacteriocins in safer hosts, obtention of multibacteriocinogenic strains with wider antimicrobial spectra, and the transfer of antimicrobial capabilities to LAB that are useful as starter, protective, and probiotic cultures in food (23).

Most bacteriocins from LAB are synthesized as biologically inactive precursors or prepeptides containing an N-terminal extension. The mature peptides are generally classified as lanthionine-containing lantibiotics (class I) and non-lanthionine-containing bacteriocins (class II) (10). However, a new classification scheme groups the enterococcal bacteriocins into class I (lantibiotic), class II (non-lantibiotic), class III (cyclic), and class IV (large proteins). Class II comprises the class II.1 (pediocin-like), the class II.2 (leaderless), and the class II.3 (non-pediocin-like) enterocins (16). The N-terminal extensions of most bacteriocins are of the so-called double-glycine type (leader sequence) and are cleaved off concomitantly with their export across the cytoplasmic membrane by dedicated ATP-binding cassette transporters and their accessory proteins (25). However, some class II bacteriocins contain N-terminal extensions of the so-called Sec type (signal peptide), which functions as a targeting and recognition signal and which is proteolytically cleaved concomitantly with bacteriocin externalization by the Sec pathway (5, 22, 26, 49). This pathway is a universally conserved protein translocation system that translocates unfolded proteins across the cell membrane via a protein-conducting pore formed by the SecYEG complex and a molecular motor, the ATPase SecA (35). Enterocins such as enterocin L50 (L50A and L50B), enterocin Q, and enterocin EJ97 are synthesized without N-terminal leader sequences and may represent a new class of bacteriocins with a novel secretion mechanism (7, 16, 40).

Hiracin JM79 (HirJM79) is a Sec-dependent class II bacteriocin produced by Enterococcus hirae DCH5 isolated from wild mallard ducks (Anas platyrhynchos). The HirJM79 structural gene (hirJM79) encodes a 74-amino-acid prepeptide containing a 30-amino-acid signal peptide (SP_HirJM79) and a 44-amino-acid mature bacteriocin (42). The broad antimicrobial activity of HirJM79 against food-borne pathogenic bacteria such as Listeria monocytogenes has attracted interest for its production by heterologous hosts. This study reports the cloning, production, and secretion of HirJM79, using different expression vectors and strains of LAB and Pichia pastoris as the production hosts.

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Bacterial strains, plasmids, growth conditions, and antimicrobial activity assays.
The LAB strains and plasmids used in this work are listed in Table 1. E. hirae DCH5, the HirJM79 producer, was isolated from the intestinal content of mallard ducks (Anas platyrhynchos) (41). Enterococci were grown in MRS broth (Oxoid Ltd., Basingstoke, United Kingdom) at 32°C. Lactococcal strains and Lactobacillus sakei Lb790 were propagated at 32°C in M17 broth (Oxoid) supplemented with 0.5% (wt/vol) glucose (GM17). For cell dry weight determinations, 10-ml samples of culture were centrifuged at 12,000 x g at 4°C for 10 min, the supernatant was discarded, and the pellet was washed and resuspended in 1 ml of distilled water with 0.85% NaCl (wt/vol). The samples were filtered under vacuum conditions through a preweighed 0.20-µm-pore-size filter (Whatman Ltd., Maidstone, United Kingdom), dried in an oven at 100°C until weight was constant, and accurately weighed. A correlation between cell dry weight and optical density at 600 nm (OD₆₀₀) was determined and used to calculate biomass concentrations. Transformants of Lactobacillus lactis, L. sakei Lb790, Enterococcus faecalis JH2-2, and E. faecium L50/14-2 were selected with 5 µg/ml of chloramphenicol (Sigma Chemical Co., St. Louis, MO). An EasySelect Pichia expression kit, the expression vector pPICZA, the wild-type P. pastoris X-33 host, and zeocin were obtained from Invitrogen Life Technologies (Madrid, Spain). Escherichia coli JM109 competent cells (Promega, Madison, WI) were propagated in Luria-Bertani (LB) broth (Sigma) at 37°C, while P. pastoris X-33 was grown in a yeast extract-peptone-dextrose (YPD) medium (Invitrogen) at 30°C. E. coli JM109 derivatives carrying pPICZA were selected on LB plates with zeocin (25 µg/ml). The P. pastoris X-33-transformed cells were selected on YPD plates with zeocin (100 µg/ml) and sorbitol (1 M) at 30°C for 3 to 10 days. The P. pastoris X-33TH clone was grown in buffered methanol minimal (BMM) medium (1.34% yeast nitrogen base [YNB], 4 x 10^–5% biotin, 100 mM potassium phosphate [pH 6], 0.5% methanol), and BMMY (BMM with 1% yeast extract and 2% peptone) at 30°C to induce the production of HirJM79. Cell-free culture supernatants were obtained by centrifugation of the grown cultures at 12,000 x g at 4°C for 10 min, adjusted to pH 6.2 with 1 M NaOH, filtered through 0.20-µm-pore-size filters (Whatman), and stored at –20°C until use. The antagonistic activity of individual colonies was screened by the stab-on-agar test, while the antimicrobial activity of cell-free culture supernatants was screened by the agar diffusion test (ADT) and, when stated, by the microtiter plate assay (MPA) (7). With the MPA, the growth inhibition of the sensitive culture was measured spectrophotometrically at 620 nm with a microtiter Labsystems iEMS plate reader (Labsystems, Helsinki, Finland). One bacteriocin unit was defined as the reciprocal of the highest dilution of the bacteriocin that caused 50% growth inhibition (50% of the control culture without bacteriocin). Recombinant cultures of L. lactis NZ9000 were induced for the production of HirJM79 when they had reached an OD₆₀₀ of 0.5, using a 4 x 10³-fold-diluted supernatant of L. lactis BB24 (the nisin A [NisA] producer) at a final concentration of 10 ng/ml.

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TABLE 1. Bacterial strains and plasmids used in this study

Basic genetic techniques and enzymes.
Total genomic DNA from E. hirae DCH5 was isolated by using a Wizard DNA purification kit (Promega, Madison, WI). Plasmid DNA isolation was performed with a QIAprep Spin miniprep kit (Qiagen, Hilden, Germany) or a High Pure plasmid isolation kit (Roche Molecular Biochemicals, Mannheim, Germany), as described by the manufacturer, with the addition of lysozyme (40 mg/ml) and mutanolysin (500 U/ml). All DNA restriction enzymes were from New England BioLabs (Beverly, MA) or Roche Molecular Biochemicals and were used as recommended by the supplier. Ligations were performed with T4 DNA ligase (Roche). Ligation mixtures were used to transform electrocompetent cells of Lactobacillus lactis, L. sakei Lb790, E. faecium L50/14-2, and E. faecalis JH2-2 with a Gene Pulser and a pulse controller apparatus (Bio-Rad Laboratories, Hercules, CA), as described previously (1, 27). Competent P. pastoris X-33 cells were obtained as recommended (Invitrogen), and electroporation of competent cells was performed as described previously (21). The proper clones were checked by a bacteriocinogenicity test and by PCR and sequencing of the inserts.

PCR amplification and nucleotide sequencing.
Oligonucleotide primers were obtained from Sigma-Genosys Ltd. (Cambridge, United Kingdom). PCR amplifications of inserts were performed in 50-µl reaction mixtures containing 1 to 3 µl of purified DNA, 70 pmol of each primer, and 1 U of Platinum Taq DNA polymerase (Invitrogen, Madrid, Spain). Samples were subjected to an initial cycle of denaturation (97°C for 2 min), followed by 35 cycles of denaturation (94°C for 45 s), annealing (50 to 62°C for 30 s), and elongation (72°C for 15 s to 3 min), and a final extension step at 72°C for 7 min in a DNA thermal cycler (Techgene; Techne, Cambridge, United Kingdom). The resulting PCR fragments were analyzed by electrophoresis in 2% agarose (Pronadisa, Madrid, Spain) gels, with a Gel Doc 1000 documentation system (Bio-Rad). The PCR-generated fragments were purified by a QIAquick PCR purification kit (Qiagen) or a QIAquick gel extraction kit (Qiagen) before they were cloned into the vectors and used for nucleotide sequencing. Nucleotide sequencing of purified PCR products was done with an ABI PRISM BigDye Terminator cycle sequence reaction kit and an ABI PRISM model 377 automatic DNA sequencer (Applied Biosystems, Foster City, CA) at a DNA sequencing service (Sistemas Genómicos, Valencia, Spain).

Recombinant plasmids for heterologous production of HirJM79 in LAB.
In a previous work, derivatives of plasmids pMG36c, pJPH1 (hirJM79), and pJPH2 (hirJM79 plus hiriJM79) carrying the structural gene and the structural-plus-immunity gene, respectively, of the HirJM79 produced by E. hirae DCH5 were constructed for the constitutive production of HirJM79 in L. lactis (42). The control and recombinant plasmids were reisolated from Lactococcus lactis IL403 and then transferred to Lactobacillus sakei Lb790, E. faecium L50/14-2, and E. faecalis JH2-2 by electroporation. The plasmid pNZ8048, containing the inducible P_NisA promoter, was also used for cloning the hirJM79 structural gene, with or without its putative hiriJM79 immunity gene. The primers and inserts used for the construction of the pNZ8048-derived plasmids are listed in Table 2. The primers HNZ-F and HPJE-R were used for PCR amplification, from fragment THi (a 711-bp fragment containing the hirJM79 and hiriJM79 genes), of a 250-bp BspHI-HindIII fragment (insert indHE) containing the hirJM79 gene. The primers HNZ-F and HPJEI-R were used for PCR amplification, from the same DNA target, of a 503-bp BspHI-HindIII fragment (insert indHEi) containing the hirJM79 and hiriJM79 genes. The fragments indHE and indHEi were digested with the indicated restriction enzymes and inserted into pNZ8048 cut with the enzymes NcoI-HindIII. The ligation mixtures were used to transform competent L. lactis subsp. cremoris NZ9000 cells. The proper clones, containing pNZH3 (hirJM79) or pNZH4 (hirJM79 plus hiriJM79), were checked with a bacteriocinogenicity test and with PCR and sequencing of the inserts.

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TABLE 2. Primers and PCR products used in this study

Heterologous production of HirJM79 in P. pastoris.
The expression vector used in this work was the plasmid pPICZA. The primers and inserts used for the construction of the recombinant plasmids are listed in Table 2. The primers PJHA-F and PJHA-R were used for PCR amplification from the fragment THi of a 170-bp XhoI-XbaI fragment (insert PH5) carrying the factor secretion signal peptide without the Glu-Ala spacer adjacent to the Kex2 protease cleavage site, fused to mature HirJM79 (hirJM79 without its signal peptide). The fragment PH5 was digested with the above-mentioned restriction enzymes, and the resulting 154-bp XhoI-XbaI-cleaved fragment was ligated into the pPICZA plasmid, digested with the same enzymes, to generate plasmid pPICH5. Competent E. coli JM109 cells were transformed with pPICH5, and the resulting transformants were confirmed by PCR amplification and sequencing. Purified pPICH5 was linearized with SacI and used to transform competent P. pastoris X-33 cells, in which the presence of the integrated pPICH5 genes was confirmed by a bacteriocinogenic test and by PCR and sequencing of the inserts.

Production of specific anti-HirJM79 polyclonal antibodies and ELISA.
The peptide fragment HJP3 (NH₂-CNHGPWAPRR-COOH), derived from the C-terminal amino acid sequence of HirJM79, was selected as the antigen for the generation of antibodies of predetermined specificity against HirJM79. The synthetic peptide HJP3 was synthesized by Invitrogen Ltd. (Paisley, Scotland, United Kingdom), with a peptide purity of >95%. The peptide HJP3 was conjugated to the keyhole limpet hemocyanin (KLH) carrier protein as an HJP3-KLH conjugate, 1:2 (wt/wt), using the components of an Imject maleimide-activated mariculture KLH kit (Perbio Science, Rockford, IL) as the immunogen. Rabbits (New Zealand White females) were immunized with HJP3-KLH, as described previously (20). Serum was obtained from blood samples incubated overnight at 4°C, centrifuged at 1,000 x g at room temperature for 15 min, and stored at –20°C until use. The enzyme-linked immunoassay (ELISA) procedures for antiserum titration and the determination of antiserum specificity and sensitivity were performed as described previously (20, 39). A noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) was developed to detect and quantify HirJM79 in the supernatants of producer strains. Briefly, wells of flat-bottomed polystyrene microtiter plates (Maxisorp, Nunc, Roskilde, Denmark) were coated overnight (4°C) with different concentrations of pure HirJM79 or with supernatants from E. hirae DCH5 or the recombinant LAB and the P. pastoris hosts. After the anti-HJP3-KLH serum and the goat anti-rabbit immunoglobulin G peroxidase conjugate (Cappel Laboratories, West Chester, PA) were added, bound peroxidase was determined with 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS; Sigma) as the substrate by measuring the absorbance of the wells at 405 nm with a Labsystems iEMS reader (Labsystems).

Purification of HirJM79 and mass spectrometry analysis.
The HirJM79 produced by P. pastoris X-33TH was purified as previously described (21). Briefly, supernatants from 400-ml cultures grown in BMMY medium were subjected to precipitation with ammonium sulfate, applied to gel filtration PD-10 columns, and further subjected to cation exchange (SP Sepharose Fast Flow) and hydrophobic interaction (Octyl Sepharose CL-4B) chromatography, followed by a reverse-phase chromatography step in a C₂-to-C₁₈ column (PepRPC HR 5/5) integrated in a reverse-phase fast-performance liquid chromatography (RP-FPLC) system. All chromatographic columns and supporting gels were from Amersham Biosciences Europe GmbH (Cerdanyola, Spain). Purified fractions from the last RP-FPLC step were subjected to matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, as described previously (23).

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Cloning and production of HirJM79 in heterologous LAB.
When derivatives of the plasmids pMG36c, pJPH1 (hirJM79), and pJPH2 (hirJM79 plus hiriJM79) were used to transform the nonbacteriocinogenic hosts Lactococcus lactis subsp. lactis IL1403 and Lactobacillus sakei Lb790, resistant to HirJM79 (Hir^r), the supernatants of the recombinant strains showed antagonistic activity against the indicator strain E. faecium T136. When the plasmid pJPH2 was used to transform E. faecalis JH2-2 and E. faecium L50/14-2, sensitive to HirJM79 (Hir^s), the strains produced halos of inhibition due to HirJM79 that were similar to or even greater than those produced by the Hir^r recombinants (results not shown). Similarly, when the pJPH1 and pJPH2 plasmids were transferred to L. lactis subsp. lactis DPC5598 and L. lactis subsp. cremoris NZ9000 (Hir^r), all recombinant derivatives were active against E. faecium T136 (Hir^s and NisA^s) (Fig. 1A), while the L. lactis DPC5598 derivatives also showed antagonistic activity against L. lactis MG1363 (Hir^r and NisA^s) as the indicator strain (Fig. 1B). When derivatives of the plasmids pNZ8048, pNZH3 (hirJM79), and pNZH4 (hirJM79 plus hiriJM79) were transferred to L. lactis NZ9000, all recombinant cultures containing either pNZH3 or pNZH4 displayed antimicrobial activity against E. faecium T136 after induction with NisA (Fig. 1C).

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FIG. 1. Antimicrobial activity of LAB cultures as determined by the ADT. (A) Supernatants of 1, L. lactis IL1403(pMG36c); 2, L. lactis IL1403(pJPH1); 3, L. lactis IL1403(pJPH2); 4, L. lactis NZ9000(pMG36c); 5, L. lactis NZ9000(pJPH1); 6, L. lactis NZ9000(pJPH2); 7, L. lactis DPC5598(pMG36c); 8, L. lactis DPC5598(pJPH1); 9, L. lactis DPC5598(pJPH2); 10, E. faecium T136; 11, E. hirae DCH5; 12, E. faecium P13, using E. faecium T136 as the indicator strain. (B) Supernatants of 1, L. lactis DPC5598(pMG36c); 2, L. lactis DPC5598(pJPH1); 3, L. lactis DPC5598(pJPH2); 4, L. lactis BB24; 5, E. hirae DCH5; 6, E. faecium P13, using L. lactis MG1363 as the indicator strain. (C) Supernatants of 1, L. lactis NZ9000(pNZ8048) before induction; 2, L. lactis NZ9000(pNZH3) before induction; 3, L. lactis NZ9000(pNZH4) before induction; 4, L. lactis NZ9000(pNZ8048) after induction; 5, L. lactis NZ9000(pNZH3) after induction; 6, L. lactis NZ9000(pNZH4) after induction; 7, E. hirae DCH5; 8, E. faecium T136, using E. faecium T136 as the indicator strain.

The heterologous extracellular production of HirJM79 by all recombinant strains was further quantified by specific anti-HirJM79 antibodies with an NCI-ELISA (Table 3). All L. lactis strains produced larger quantities of HirJM79 than E. hirae DCH5. The production of HirJM79 by derivatives of L. lactis IL1403, carrying plasmids pJPH1 and pJPH2, was 5.5- to 7.8-fold higher, respectively, while the production of HirJM79 by derivatives of L. lactis NZ9000, transformed with the same plasmids, was 3.0- to 3.9-fold higher, respectively, than the production of HirJM79 by E. hirae DCH5. The production of HirJM79 by derivatives of L. lactis NZ9000 carrying the pNZH3 and pNZH4 recombinant vectors was also 4.7- to 8.5-fold higher, respectively, than that of E. hirae DCH5. The production of HirJM79 was higher in the L. lactis strains carrying the plasmids pJPH2 and pNZH4 than in those transformed with pJPH1 and pNZH3. However, the antimicrobial activities and the specific antimicrobial activities of these L. lactis transformants was lower than expected from their production of HirJM79. The transformation of L. lactis DPC5598, a NisA producer strain, with the recombinant pJPH1 and pJPH2 plasmids permitted the coproduction of HirJM79 and NisA, as determined by ADT and an NCI-ELISA (Fig. 1A and B). The production of HirJM79 by L. lactis DPC5598(pJPH1) and L. lactis DPC5598(pJPH2) was 4.1- to 5.6-fold greater, respectively, than that by E. hirae DCH5. The production of NisA by L. lactis DPC5598(pMG36c) and by its two HirJM79⁺ derivatives was similar but lower (41% to 53%) than that of L. lactis BB24 (Table 3). The Lactobacillus sakei strain Lb790(pJPH1) and the L. sakei Lb790(pJPH2) transformants also produced larger quantities of HirJM79 than E. hirae DCH5 did, although the antimicrobial activities and the specific antimicrobial activities of both derivatives were lower than expected from their production of HirJM79. The E. faecium L50/14-2(pJPH2) and E. faecalis JH2-2(pJPH2) strains were slightly greater producers of HirJM79 than E. hirae DCH5, although they displayed a higher antimicrobial activity (Table 3).

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TABLE 3. Bacteriocin production and antimicrobial activity of supernatants from recombinant strains

Cloning and heterologous production of HirJM79 by P. pastoris.
The P. pastoris X-33TH clone was selected for its high level of antagonistic activity from among different P. pastoris X-33 derivatives carrying the integrated pPICH5 plasmid, by using the stab-on-agar test (results not shown). The heterologous production of HirJM79 by P. pastoris X-33TH was further evaluated and quantified by an ADT, an MPA, and an NCI-ELISA. As shown in Fig. 2, the supernatants of P. pastoris X-33TH grown in BMM medium produced smaller halos of inhibition (Fig. 2A) than the supernatants of the same culture grown in the BMMY complex medium (Fig. 2B). Moreover, the antimicrobial activity of cultures grown in BMMY medium lasted longer than that of cultures grown in BMM medium. The results shown in Table 4 also indicate that the maximum production of HirJM79 during the growth of P. pastoris X-33TH in BMM medium represents a 5.0-fold increase over the production by E. hirae DCH5, but its maximum antimicrobial activity was 33%, and its maximum specific antimicrobial activity was 9% of the HirJM79 produced by E. hirae DCH5. However, the maximum production of HirJM79 by P. pastoris X-33TH grown in BMMY medium was 2.0-fold higher, and its maximum antimicrobial activity was 76% of the production and activity of the HirJM79 produced by E. hirae DCH5. The maximum specific antimicrobial activity of the HirJM79 produced by P. pastoris X-33TH grown in BMMY medium was 42% of the HirJM79 produced by E. hirae DCH5. Purification of the antimicrobial peptide produced by P. pastoris X-33TH grown in BMMY medium permitted a 2,804-fold increase of its specific activity and a 9% recovery of the initial antimicrobial activity (Table 5). MALDI-TOF mass spectrometry analysis of the purified antagonistic peptide of P. pastoris X-33TH showed a major peak at 5,093.2 Da, suggesting that the peptide was purified to homogeneity (results not shown).

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FIG. 2. ADT for detection of the HirJM79 antimicrobial activity against E. faecium T136. Wells contain supernatants of P. pastoris X-33TH grown in (A) the BMM medium or (B) the BMMY complex medium after 0 (d), 4 (e), 6 (f), 8 (g), 10 (h), 12 (i), and 24 (j) h of incubation. Supernatants of P. pastoris X-33 cultures grown in BMM or BMMY medium for 0 (a), 6 (b), and 12 (c) h were used as negative controls. Supernatants of E. hirae DCH5, not diluted (l), 4-fold diluted (m), and 16-fold diluted (n), were used as positive controls, and the supernatant of E. faecium T136 (k) was used as a negative control.

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TABLE 4. Production and antimicrobial activity of HirJM79 from supernatants of Pichia pastoris X-33TH grown in different mediaa

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TABLE 5. Purification of the antimicrobial activity of P. pastoris X-33TH

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The cloning and production of HirJM79, a Sec-dependent bacteriocin produced by E. hirae DCH5, have been evaluated in heterologous LAB strains and P. pastoris. All recombinant LAB strains displayed antagonistic activity (Fig. 1), confirming that hirJM79 is the minimum requirement for the production of biologically active HirJM79 in bacteriocin-resistant hosts (42). Contrary to many studies that report the heterologous expression of antimicrobial peptides by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analyses, the use of specific anti-HirJM79 antibodies and an NCI-ELISA permitted the detection and quantification of HirJM79 production by recombinant LAB strains. The production of HirJM79 by all LAB strains was higher than that by E. hirae DCH5 (Table 3), whereas the production of HirJM79 in L. lactis IL1403 was larger than that in L. lactis NZ9000 transformed with the same vectors. Differences in the production of bacteriocins by lactococcal strains have been reported previously (23, 28, 37, 38) and may reflect unknown metabolic differences between L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000. The increased antimicrobial activity of L. lactis NZ9000, transformed with pNZH3 and pNZH4, may be ascribed to the short induction time for HirJM79 production, which most probably prevents HirJM79 from attaching to cell walls, forming aggregates, and/or undergoing proteinase degradation.

Coexpression of the hirJM79 and hiriJM79 genes increased the production of HirJM79 by all lactococcal hosts. Increased HirJM79 production may be explained by assuming that L. lactis is relatively resistant to HirJM79, but it may endure more of the bacteriocin when it expresses the HirJM79 product. Indeed, bacteriocin producers are protected from their own bacteriocin by the concomitant expression of a cognate immunity protein. These proteins act either by affecting bacteriocin aggregation and pore formation or by disturbing the interaction between the bacteriocin and the membrane-located bacteriocin receptor (23). Coexpression of the structural and immunity genes also increased the heterologous production of enterocin P (EntP), a Sec-dependent bacteriocin produced by E. faecium P13 in lactococci (23). The production of HirJM79 also depended on the lactococcal vector used (Table 3). The enhanced HirJM79 production in L. lactis NZ9000, using the nisin-inducible constructs, may be due to copy number differences between pNZ8048 and pMG36c but, more likely, is caused by the different promoters used to drive gene expression (13, 32).

Recombinant L. lactis strains able to coexpress NisA and HirJM79 were constructed by using L. lactis subsp. lactis DPC5598, a plasmid-free derivative of an industrial strain producer of NisA (45). The low production of HirJM79 by the recombinant L. lactis DPC5598 derivatives compared to that achieved by L. lactis IL1403 transformed with the same plasmids may be attributed to the production of more than one bacteriocin in multibacteriocinogenic hosts or to the genomic or metabolic differences between the strains or, most probably, to the higher proteinase activity of L. lactis DPC5598 (23). The low production of NisA by the L. lactis DPC5598 transformants compared to that of L. lactis BB24 may also be ascribed to unknown genetic and/or metabolic differences between the strains. Since NisA is particularly active against clostridia and their spores and HirJM79 is a wide-spectrum bacteriocin with antilisterial activity, these recombinant strains with antagonistic synergy would be of biotechnological interest for the food industry. The production of HirJM79 was higher in Lactobacillus sakei Lb790 than in E. faecium L50/14-2 and E. faecalis JH2-2, although the E. faecium L50/14-2(pJPH2) and the E. faecalis JH2-2(pJPH2) hosts produced slightly larger amounts of HirJM79 than E. hirae DCH5. E. faecium L50/14-2(pJPH2) was also a coproducer of EntP and HirJM79. These results indicate the functionality of the SP_HirJM79 to drive the efficient secretion of HirJM79 in LAB strains. The secretory production of proteins provides advantages compared to cytosolic production, and protein secretion is a preferred means of protein expression for LAB as vehicles for the delivery of biologically active molecules (14).

However, the antimicrobial activity and the specific antimicrobial activity of the HirJM79 produced by most of the heterologous LAB were lower than that expected from their production of HirJM79 (Table 3). Since purification of HirJM79 from L. lactis IL1403(pJPH1) and E. hirae DCH5 yielded, according to MALDI-TOF analysis, fragments of identical molecular masses (42), this suggests that HirJM79 is processed and exported adequately out of the L. lactis cells. Thus, it is possible to speculate that disulfide bond formation (DSB) in HirJM79 (between Cys 10 and Cys 15) is not performed adequately by lactococci. DSB is a universally conserved mechanism for stabilizing extracytoplasmic proteins, carried out by thiol-disulfide oxidoreductases, which catalyze the formation, disruption, or isomerization of disulfide bonds in proteins (31). However, the function of the thiol-disulfide oxidoreductases in most bacteria remains largely unexplored (33). Although L. lactis has the capacity to synthesize proteins containing DSB, the genome sequencing of several strains has not revealed lactococcal homologs of genes involved in DSB in other bacteria (17). Alternatively, the bacteriocin could undergo conformational modifications, rendering less active extracellular HirJM79 forms. It may also happen that the posttranslational processing of pre-HirJM79 to active HirJM79 may be less efficient in Lactococcus than in Enterococcus, as it may occur with bacteriocins heterologously produced by Lactobacillus and Pediococcus (15, 28).

A higher aggregation of HirJM79 and/or a higher proteolytic activity of the lactococcal bacteriocin-producing hosts may also reflect the lower antimicrobial activity of the HirJM79 produced by the lactococcal strains and Lactobacillus sakei Lb790. EntP, a Sec-dependent class II.1 bacteriocin closely related to HirJM79, tends to form aggregates that reduce its biological activity (20). However, E. faecium L50/14-2(pJPH2) and E. faecalis JH2-2(pJPH2) produce HirJM79 with a higher specific antimicrobial activity than that of E. hirae DCH5. Since the HirJM79 produced by E. hirae DCH5 is identical to the bacteriocin T8 produced by E. faecium T8 and to the bacteriocin 43 produced by E. faecium VRE82 and shows homology with the bacteriocin RC714 produced by E. faecium RC714 and the bacteriocin 31 produced by E. faecalis Y1717 and with the EntP produced by E. faecium P13 (42), it is possible to speculate that E. faecium and E. faecalis behave as more efficient hosts for the production of HirJM79 than E. hirae does. Nevertheless, the HirJM79 produced by the recombinant lactococcal hosts or added as cell-free cultures may be attractive for biotechnological applications. The use of "food grade" organisms as producing strains may also provide a means by which the potential benefits of antimicrobial compounds can be exploited in food.

The production of bacteriocins by heterologous hosts may be based on the expression of native biosynthetic genes, the exchange of leader peptides, and/or the dedicated secretion and processing systems (ATP-binding cassette transporters) or the fusion of mature bacteriocins to signal peptides that act as secretion signals (23). As reported in this work, the high-level production of HirJM79 by LAB hosts suggests that the production of this bacteriocin by the Sec pathway is efficient in Lactococcus lactis, Lactobacillus sakei, E. faecium, and E. faecalis. It remains unclear why most LAB bacteriocins have a dedicated processing and secretion system when they can access the Sec pathway if it is provided with an appropriate signal peptide. Fusions between the SP_HirJM79 and the mature part of other bacteriocins may allow LAB strains to secrete bacteriocins in the absence of specific immunity and secretion proteins. Chimeras of pediocin PA-1 (PedA-1) and enterocin A (EntA) fused to the signal peptide of EntP (SP_EntP) have permitted the production and functional expression of these bacteriocins in L. lactis (37, 38). However, further efforts to improve the antimicrobial activity of the HirJM79 produced by LAB would be desirable. Construction of the L. lactis strain deficient in both of its major proteases, intracellular (ClpP) and extracellular (HtrA), may show its usefulness for the production of more stable heterologous peptides (9). The production of peptides with DSB is also a challenge for the development of LAB strains engineered for the expression of biologically active peptides (17).

Heterologous expression systems for the production and secretion of bacteriocins are being developed in bacteria. However, yeasts have not been fully exploited as alternative hosts for the production of bacteriocins. In this work, the heterologous production and functional expression of HirJM79 in P. pastoris, a methylotrophic yeast (29), have been achieved. The pPICZA plasmid was selected as the expression vector because it contains the Saccharomyces cerevisiae alpha mating factor (prepro) signal sequence to target fused proteins to the P. pastoris secretory pathway, a methanol-inducible promoter, and the AOX1 region that allows integration of the vector into the 5' AOX1 locus of the P. pastoris X-33 genome. The high production of HirJM79 in the BMM medium compared to that in the BMMY complex medium may be ascribed to a better antigen epitope recognition of HirJM79 by the anti-HirJM79 antibodies in the former than in the latter medium. The activity of neutral proteases may also be responsible for the reduced antimicrobial activity of supernatants of P. pastoris X-33TH grown in BMM compared to that of supernatants grown in BMMY medium (Table 4). Nevertheless, the production of HirJM79 by P. pastoris X-33TH grown in BMMY medium was higher than that in the enterococci, including that of E. hirae DCH5, but not in lactobacilli and lactococci, although its antimicrobial activity and specific antimicrobial activity were lower than expected from their production of HirJM79 (Table 4). Since purified HirJM79 from P. pastoris X-33TH (results not shown) and from E. hirae DCH5 (41) show identical molecular masses, this suggest that posttranslational events typical of yeasts (12) have not occurred. Thus, it seems that the activity of neutral proteases may be also responsible for the loss of antimicrobial activity of supernatants of P. pastoris X-33TH grown in BMMY medium. Although other bacteriocins such as pediocin PA-1 (2, 44) and plantaricin 423 (48) have been produced by recombinant S. cerevisiae and P. pastoris hosts, no inhibitory activity was detected in their supernatants. Currently, only EntP (21) and HirJM79 stand as biologically active bacteriocins produced by P. pastoris. The production and antimicrobial activity of HirJM79 in the supernatants of P. pastoris X-33TH may facilitate future biotechnological applications of this bacteriocin as a natural antimicrobial peptide in food, pharmaceutical, veterinary, and medical applications. However, before large-scale applications of this bacteriocin are attempted, further strategies for the optimal production of HirJM79 by the recombinant P. pastoris hosts should be developed.

 
We thank Paul Ross (Teagasc Dairy Products Research Centre, Moorepark, Fermoy, County Cork, Ireland) for providing L. lactis DPC5598, Jan Kok (Department of Genetics, University of Groningen, The Netherlands) for supplying plasmid pMG36c, and Rosa del Campo (Departamento de Microbiología, Hospital Universitario Ramón y Cajal, Madrid, Spain) for supplying the strain JH2-2. The help of Jorge Gutiérrez is also recognized.

This work was supported in part by grants AGL2003-01508 and AGL2006-01042 from the Ministerio de Educación y Ciencia (MEC) and by grant S-0505/AGR/0265 from the Comunidad de Madrid (CAM), Spain.

J. Sánchez holds a fellowship from the Ministerio de Educación, Cultura y Deporte (MECD), Spain. J. Borrero holds a research contract from the CAM, and A. Basanta received an FPI fellowship from the CAM, Spain. B. Gómez-Sala holds a research contract from Innaves S.A. (Vigo, Spain).

 
* Corresponding author. Mailing address: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, Avenida Puerta de Hierro, s/n, 28040 Madrid, Spain. Phone: 34-91-3943752. Fax: 34-91-3943743. E-mail: ehernan{at}vet.ucm.es

Published ahead of print on 29 February 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Applied and Environmental Microbiology, April 2008, p. 2471-2479, Vol. 74, No. 8
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