INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Lactic acid bacteria (LAB) and the bacteriocins many of them produce have an important role as future food biopreservatives due to increasing consumer awareness of the potential risks derived not only from food-borne pathogens, but also from the artificial chemical preservatives used to control them. The practical application of bacteriocin-producing LAB in biocontrol may find some limitations, such as a narrow antimicrobial spectrum, low-level or unstable production, and the inability to grow in foods in which the bacteriocin(s) produced would be particularly effective (1). However, in recent years there has been a rapid growth in understanding of the genetics of many of these industrially important organisms, and improved techniques for their genetic manipulation have provided the tools to overexpress bacteriocins, to engineer bacteriocin variants with improved properties (7, 24), and to transfer bacteriocin biosynthesis genes to other species (2, 4, 22, 40).

An attractive approach to achieving heterologous production of antimicrobial peptides is based on the significant amino acid homologies shared by the leader peptides, and also by the dedicated transporters, of most class II bacteriocins of LAB, some lantibiotics, and also colicin V produced by Escherichia coli (15, 16, 41). This class of leader peptide is cleaved at a specific processing site adjacent to two conserved glycine residues located at positions 1 and 2 (15). Gly₂ is 100% conserved in different class II bacteriocins, underlining the importance of a glycine at this position. Moreover, the introduction of mutations affecting this residue in colicin V and lactacin F has the effect of generating mutants that are able to express, but not to export, the respective bacteriocins (9, 12). The role of the N-terminal domain of the ATP-binding cassette (ABC) transporter of lactococcin G (LagD) in proteolytic cleavage of the leader sequence of lactococcin Ga was convincingly demonstrated by Havarstein et al. (16) in in vitro studies employing a purified N-terminal polypeptide (150 amino acids). Correct cleavage at the consensus processing site indicated that precursor bacteriocins with double-glycine leader peptides are processed, concomitantly with export, by a family of dedicated ABC transporters (16).

Evidence that double-glycine leader peptides serve as recognition signals for the dedicated ABC transporters was provided by van Belkum et al. (40). In their work, the leader peptides of leucocin A, lactococcin A, and colicin V were fused to divergicin A, a bacteriocin produced by Carnobacterium divergens and secreted via the general secretory pathway (43). In the homologous hosts, the various leader peptides were able to direct the secretion of divergicin by using their dedicated transport machinery (40). Colicin V secretion was also achieved, although less efficiently, in Lactococcus lactis by using the lactococcin A secretion machinery when the colicin V peptide was fused to the leucocin A leader peptide. Allison et al. (2) have also shown that both peptides of the two-component lactacin F complex can use the secretion machinery of Carnobacterium piscicola LV17, a strain that produces carnobacteriocins A, BM1, and B2.

Pediocin PA-1 is a wide-spectrum bacteriocin produced by some Pediococcus acidilactici strains of meat origin (3, 13, 17, 27, 30) and Lactobacillus plantarum WHE92 (8). We have recently reported the heterologous production of this bacteriocin in L. lactis by introducing a vector containing an in-frame fusion between sequences encoding the lactococcin A leader and mature pediocin PA-1 (22). L. lactis IL1403 was selected as the host because it is a pediocin PA-1-resistant, plasmid-free strain that does not produce bacteriocin but that harbors chromosomal genes analogous to those encoding the lactococcin A secretion apparatus, lcnC and lcnD (35, 42). The resulting L. lactis strain was able to secrete pediocin PA-1 at a level approximately 25% of that displayed by the parental pediocin-producing P. acidilactici 347. Significant reductions in the yield of lactococcin A expressed in IL1403 have been previously described (19, 39). These observed reductions may be attributed to the low copy number of the chromosomal lcnC and lcnD gene analogs (35) and/or to the fact that the products of these genes are not identical to the equivalent lactococcin A translocatory apparatus (42), resulting in a less efficient secretion process. It has been demonstrated that bacteriocin production can increase at least 10-fold when the dedicated lcnC and lcnD genes are included in equivalent lactococcal expression systems (40, 42).

The aim of this study was to extend and improve the production levels of heterologously expressed peptides which employed the lactococcin A secretory apparatus (22). The lcnC and lcnD genes from the lactococcin A-producing strain L. lactis WM4 (35) were introduced into the pediocin PA-1-producing derivative of L. lactis IL1403, and the effect that the dedicated transport machinery had on pediocin PA-1 production levels was assessed. Heterologous expression and secretion of pediocin PA-1 in other L. lactis hosts have also been investigated, resulting in the enhanced production of pediocin PA-1 and the coproduction of the lantibiotic nisin A and pediocin PA-1.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Microbiological techniques, strains, and plasmids. Lactococcal strains and plasmids used in this study are listed in Table 1. Cultures were routinely grown in MRS (Oxoid, Unipath Ltd., Basingstoke, United Kingdom) or M17 medium (38) supplemented with 0.5% (wt/vol) glucose (GM17 medium) at 30°C without agitation. P. acidilactici 347 (30) was grown in MRS medium at 30°C without agitation. E. coli was grown in L broth (25) at 37°C on an orbital shaker. Agar plates were made by addition of 1.5% (wt/vol) agar to broth media. Antibiotics were added as selective agents when appropriate: chloramphenicol, 5 µg ml¹ for lactococci and 15 µg ml¹ for E. coli; ampicillin, 200 µg ml¹; and erythromycin, 5 µg ml¹.

1

Molecular techniques. Plasmid DNA isolation was carried out as previously described (5). Restriction enzymes and other DNA-modifying enzymes from various sources were used according to the suppliers' recommendations. Recombinant plasmids were recovered by transformation of E. coli (6) or by electroporation of L. lactis according to the method of Holo and Nes (18) with the modifications of Dodd et al. (6). Conditions used for PCR were as described by Horn et al. (21), and primers were synthesized on an Applied Biosystems DNA synthesizer (model 381A). Fragments generated for the construction of vectors were amplified with DyNAzyme  DNA polymerase (Flowgen) and cloned into pCRII (Invitrogen). For routine PCR screening of recombinant clones AmpliTaq DNA polymerase (Perkin-Elmer) was used. Confirmation of nucleotide sequences was carried out on purified plasmid DNA with an Applied Biosystems DNA sequencer (model 373A) and the manufacturer's Taq DyeDeoxy Terminator Cycle sequencing kit.

Cloning functional lcnC and lcnD genes. A 3.8-kb fragment of pNP2 DNA from L. lactis WM4 containing the lcnC and lcnD genes and upstream promoter (Fig. 1a) was amplified by PCR with the flanking primers P136 (5'-GAGGCAGTAAGTAATATTATTTTC-3') and P137 (5'-ACTCTACTGATTGCCTCTTCC-3') and cloned into pCRII (Invitrogen). The amplified genes were then subcloned, as a SacI/XbaI fragment, into pFI2058 (a derivative of the shuttle vector pTG262 that contains lcnA and lciA genes [Fig. 1b]) (22), and a mixture of the resulting recombinant plasmids, carrying all four lcn genes, was introduced into L. lactis MG1614 (plasmid free, Lcn). In order to identify derivatives containing plasmids carrying functional lcnC and lcnD genes (that had not suffered inactivating PCR-induced mistakes), colony overlays were carried out on the transformed colonies. In this way a transformant that contained the plasmid pFI2149 (Fig. 1c) and that secreted lactococcin A generating a zone of inhibition of growth of the overlying indicator strain, MG1614, was isolated. The active lcnC and lcnD genes of pFI2149 were recovered as an EcoRI fragment and cloned into the vector pIL277 to generate pFI2148 (Fig. 1d). This same fragment was cloned into the EcoRI site of pFI2126 (Fig. 1e), upstream of the L-pedA gene (hybrid lcnA/pedA gene [22]) to generate plasmid pFI2160 (Fig. 1f). Nucleotide sequence analysis of this fragment, as described above, revealed that the cloned lcnC and lcnD genes showed 100% identity to the parental genes from L. lactis WM4 and had not suffered any mutations as a result of PCR-induced mistakes.

View larger version (33K): [in this window] [in a new window]   FIG. 1.   Maps showing genes involved in lactococcin A and pediocin PA-1 production. Thick arrows show the locations and orientations of the coding regions. (a) Lactococcin A determinants carried by pNP2 (lcnC, lcnD, lcnA, and lciA genes). Small arrows above and below the map indicate the positions and directions of primers used for PCR amplification of the lcnC and lcnD genes. (b) pFI2058; (c) pFI2149; (d) pFI2148; (e) pFI2126; (f) pFI2160 containing lcnA/pedA hybrid gene L-pedA. Relevant restriction enzyme sites are as follows: S, SacI; X, XbaI; E, EcoRI.

Purification of pediocin PA-1. The bacteriocin produced by L. lactis FI9267 was purified from a 1-liter culture grown in MRS broth at 30°C to late logarithmic phase. The procedure, involving ammonium sulfate precipitation and, successively, cation exchange, hydrophobic interaction, and reverse-phase chromatography (PepRPC HR5/5 fast-performance liquid chromatography system; Pharmacia LKB, Uppsala, Sweden) was essentially as previously described (22, 28). The active fractions, obtained after hydrophobic interaction chromatography, were applied to the reverse-phase column, and the bacteriocins were eluted with a linear gradient ranging from 10 to 50% 2-propanol containing 0.1% trifluoroacetic acid. Purification steps were performed at room temperature, and the chromatographic equipment and reagents were obtained from Pharmacia and used according to the supplier instructions. The microtiter plate assay system developed by Holo et al. (19) was employed to quantify the bacteriocin activities during the purification process. One bacteriocin unit (BU) was defined as the reciprocal of the highest dilution causing 50% growth inhibition of the indicator organisms E. faecalis TAB28 (pediocin PA-1 sensitive) and L. lactis MG1614 (nisin sensitive). The reverse-phase fraction containing each pure bacteriocin was desiccated by rotary evaporation and redissolved in an equivalent volume of deionized water. The amino-terminal sequence of the purified bacteriocin was determined by Edman degradation with an automatic sequencer (model 47A; Applied Biosystems).

Specific detection of pediocin PA-1 and nisin A by ELISA. A competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was used to assess the production of pediocin PA-1 by strains used in this study. Pediocin PA-1, purified from P. acidilactici 347 (as described above), was diluted in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6) to give a concentration of 750 ng ml¹. Samples (100 µl) were incubated in 96-well polystyrene microtiter plates (Maxisorp; Nunc, Roskilde, Denmark) for 16 h at 4°C. After bacteriocin coating, the wells were washed with 0.01 M phosphate-buffered saline (PBS), pH 7.4, and blocked with PBS containing 1% ovalbumin for 1 h at 37°C. Then 50 µl of MRS culture supernatants and 50 µl of antiserum, diluted 1:250 in PBS, added, and the plates were incubated for 1 h at 37°C. The antiserum contained rabbit antibodies raised against a synthetic peptide (PH1) comprising the amino-terminal nine amino acids of pediocin PA-1 and conjugated to keyhole limpet hemocyanin (Pierce Chemical Co., Rockford, Ill.) through its carboxy-terminal cysteine residue (25a).

1

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Enhanced lactococcin A production directed by the dedicated secretory proteins LcnC and LcnD. Attempts to improve bacteriocin production efficiency in L. lactis involved two strategies for introducing the genes specifying the lactococcin A-dedicated translocatory machinery (lcnC and lcnD) into derivatives utilizing the expression system described previously (22). First, the lcnC and lcnD genes were cloned into the vector pIL277, generating pFI2148 (Fig. 1d). This plasmid is compatible with the pTG262-based vector, pFI2058, carrying the lcnA and lciA genes (Fig. 1b), allowing the two plasmids to be stably maintained in the hosts MG1614 and IL1403. In the second approach the 3.8-kb fragment carrying the lcnC and lcnD genes was cloned into pFI2058 to generate the recombinant plasmid pFI2149, which harbors all four genes of the lactococcin A gene cluster arranged in two operons as shown in Fig. 1c.

LcnC and LcnD directed secretion of pediocin PA-1. Experiments were carried out to determine whether a similar improvement could be achieved with the heterologous production of pediocin PA-1 by L. lactis. The IL1403 derivative FI9043 carries the recombinant plasmid pFI2126, which contains an in-frame fusion of sequences encoding the lactococcin A leader and the mature part of pediocin PA-1 (denoted by the genotype L-pedA [Fig. 1e]). Expression of this hybrid gene, under the control of the lcnA promoter, results in low-level production of pediocin PA-1 (22). FI9043 was transformed with the compatible plasmid pFI2148 (lcnC lcnD) (Fig. 1d) generating FI9181. Both plasmids pFI2126 and pFI2148 were also introduced into L. lactis MG1614, resulting in strain FI9180. Expression of all the genes necessary for pediocin PA-1 production was also achieved in a single replicon on the recombinant plasmid pFI2160 (Fig. 1f). This was introduced into both IL1403 and MG1614 to generate strains FI9265 and FI9263, respectively.

View larger version (86K): [in this window] [in a new window]   FIG. 2.   Plate diffusion bioassays for detection of pediocin PA-1 activity against E. faecalis TAB28 (a) and nisin A activity against L. lactis MG1614 (b). Wells contained supernatants extracted from L. lactis cultures: 1, FI9180; 2, FI9181; 3, FI9262; 5, FI5876; 6, FI9263; 7, FI9265; 8, FI9267; 9, MG1614; 10, IL1403. Well 4 contained P. acidilactici 347 supernatant.

Dual production of pediocin PA-1 and nisin A. The nisin A-producing strain Lactococcus lactis subsp. cremoris FI5876 was transformed with the recombinant plasmids constructed for the heterologous production of pediocin PA-1, as described above. Derivatives of FI5876 containing either pFI2126 (strain FI9261), pFI2126 and pFI2148 (strain FI9262), or pFI2160 (strain FI9267) were assayed for activity against the indicator organisms E. faecalis TAB28 (pediocin PA-1 sensitive; nisin resistant) and L. lactis MG1614 (pediocin PA-1 resistant; nisin sensitive) to determine whether they were able to simultaneously produce pediocin PA-1 and nisin A.

1

Bacteriocin purification. The antimicrobial peptides produced by FI9267, which achieved the highest levels of simultaneous pediocin PA-1 and nisin A activity (Table 2), were purified for further analysis (Table 3). Fractions from the first run on the reverse-phase column, which showed the highest activities against E. faecalis TAB28 and L. lactis MG1614, were collected and rechromatographed. Two absorbance peaks, the first (29% 2-propanol; peak 1) coincident with the E. faecalis TAB28 activity peak and the second (31% 2-propanol; peak 2) coincident with the L. lactis MG1614 activity peak, were observed (Fig. 3). The final specific activities of these two pure bacteriocins were approximately 1.3 × 10⁴- and 3.2 × 10⁴-fold higher, respectively, than that in the crude culture supernatant, and the recoveries were 6 and 17%, respectively (Table 3).

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Reverse-phase chromatography of pediocin PA-1 and nisin A. The amount applied to the column was obtained from a 1-liter culture of L. lactis FI9267. , activity against E. faecalis TAB28; , activity against L. lactis MG1614. TFA, trifluoroacetic acid.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Expression of the lcnA (structural) and lciA (immunity) genes is the minimum requirement for production of lactococcin A in L. lactis IL1403, which encodes translocation functions that recognize and process the pre-lactococcin A peptide (35, 42). On this basis we developed an expression system for heterologous production of pediocin PA-1 in the naturally resistant L. lactis IL1403 (22). Expression of the chimeric gene (L-pedA) encoding the lactococcin A leader and the mature part of pediocin PA-1 preceded by the upstream promoter of lcnA resulted in secretion of mature pediocin PA-1 into L. lactis FI9043 culture supernatants (22). The yield of pediocin PA-1 from this lactococcal strain was low (maximum yield attained was 270 ng ml¹) and, as with lactococcin A production in the parent strain, L. lactis WM4, the levels of activity fluctuated in different cultures.

It has been suggested that the single copies of the chromosomally encoded lcnC and lcnD gene analogs and/or the differences in nucleotide sequence between these and the dedicated lcnC and lcnD genes may be responsible for less-efficient bacteriocin secretion in IL1403 (19, 22, 35, 42). The strategy used to improve production of heterologous peptides in L. lactis, directed by the lactococcin A leader, was (i) to employ the dedicated translocatory genes and (ii) to increase the gene dose by using multicopy plasmids. Pediocin PA-1 production by FI9043 (443 ng ml¹) was approximately doubled as a result of transformation with pFI2148 (lcnC lcnD) to generate FI9181 (934 ng ml¹) (Table 2), indicating that the cloned genes are more effective at mediating pediocin PA-1 translocation in this strain. This enhanced level of production still represented only half that of the natural pediocin PA-1 producer P. acidilactici 347. A further yield increase was achieved when the cloned lcnC and lcnD genes were carried on the same plasmid as the hybrid L-pedA gene on pFI2160 (Fig. 1f), and L. lactis IL1403 carrying this recombinant plasmid (FI9265) attained pediocin PA-1 levels approaching those of P. acidilactici 347 (Table 2).

One reason for this increased yield is that all the genes required for heterologous production of pediocin PA-1 were carried on the same plasmid, pFI2160 (Fig. 1f), derived from the lactococcal plasmid pSH71, which is maintained at 50 to 60 copies per cell (11). Thus, the observed higher production levels could be attributed to the increased lcnC and lcnD gene doses compared to those for cells carrying the equivalent genes on the low-copy-number plasmid pFI2148 (six to nine copies per cell [33]). In a study carried out by Chikindas et al. (4), a strain of L. lactis IL1403 containing the four ped determinants cloned into a lactococcal vector was able to produce pediocin PA-1 at low levels. This was directed by its own pedA-encoded leader and involved the dedicated pedC and pedD translocation genes. Enhanced pediocin PA-1 production was achieved by increasing the copy number of the plasmid-borne ped operon. The maximum yield reported by these workers was approximately 50% of the pediocin PA-1 level attained by strains generated in this work. This indicates that, in IL1403, lactococcin A-directed secretion of pediocin PA-1 is more efficient that the equivalent process directed by the pediocin PA-1 translocatory machinery.

It is noteworthy that the yields of lcnC- and lcnD-mediated translocation of both lactococcin A and pediocin PA-1 were also influenced by the particular lactococcal strain employed as a production host. A comparison of L. lactis strains IL1403, MG1614, and FI5876, carrying the equivalent recombinant plasmids for either lactococcin A or pediocin PA-1 production, revealed that IL1403 consistently achieved higher activity levels (Table 1). For pediocin PA-1 production levels, which have been quantified, IL1403 produced at least four times the yield of the other two host strains (Table 2). This observed yield variation may reflect metabolic differences between Lactococcus lactis subsp. lactis (IL1403) and L. lactis subsp. cremoris (MG1614 and FI5876). Nucleotide sequence variation between the plasmid-borne lcnC and lcnD and chromosomal analogs has been observed (20a), and this variation may result in functional variation between the two translocation systems. Alternatively, differences in the level of expression of these analogous genes may contribute to the relatively high yields generated by IL1403. The latter possibility may be brought about by sequence divergence in the upstream promoter regions or differences in copy number control of the expression plasmid in the two hosts. A greater understanding of how host-encoded functions can influence production of heterologous peptides in L. lactis awaits further investigation of these genes at the molecular level.

In this study L. lactis strains that were able to express and secrete nisin A together with pediocin PA-1 were constructed (FI9262, FI9267) (Table 2). This represents a first step in the construction of LAB strains able to coproduce two or more well-characterized wide-spectrum bacteriocins. Nisin, the only antimicrobial peptide currently licensed for use as a food additive, is particularly active against clostridia and their spores, while pediocin PA-1, because of its anti-Listeria activity, is a candidate for future approval (34). Using pure bacteriocins as food additives is currently a controversial issue. However, employing "food-grade" organisms as production strains may provide a means by which the potential benefits of these antimicrobial compounds can be exploited (20).

The antimicrobial efficiency of a bacteriocin may be enhanced or broadened by combining it with other bacteriocins. This synergistic effect has been observed with a combination of sakacin A and nisin A, which inhibited the growth of Listeria monocytogenes much more strongly than either of the bacteriocins alone (32). Similarly, Hanlin et al. (14) reported the increased antibacterial activity of a combination of nisin A and pediocin PA-1 against several gram-positive bacteria, including strains of L. monocytogenes and clostridial strains. Recently, Mulet-Powell et al. (26) have reported the increased potency of pediocin PA-1 when used in combination with other bacteriocins, demonstrating that, in addition to nisin A, it can act synergistically with lacticin 481, lacticin B, or lacticin F.

The fact that nisin and pediocin PA-1 are completely unrelated bacteriocins may have practical consequences. The emergence of organisms resistant to class II bacteriocins has become quite common and is a potential obstacle to their application as antimicrobial agents. Rekhif et al. (29) have described mutants of L. monocytogenes that have spontaneously acquired resistance to three pediocin-like bacteriocins, mesenterocin 52, curvaticin 13, and plantaricin C19. Although these mutants displayed cross-resistance to other members of this bacteriocin class, their sensitivity to nisin had not been affected. The particular antimicrobial properties of pediocin PA-1 and nisin A and the beneficial and synergistic effects of their coproduction are features that can be exploited to extend their potential application in the food industry.