Scholarly article on topic 'Cloning strategies for heterologous expression of the bacteriocin enterocin A by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475'

Cloning strategies for heterologous expression of the bacteriocin enterocin A by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475 Academic research paper on "Veterinary science"

0
0
Share paper
Academic journal
Microb Cell Fact
OECD Field of science
Keywords
{""}

Academic research paper on topic "Cloning strategies for heterologous expression of the bacteriocin enterocin A by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475"

Jiménez et al. Microb Cell Fact (2015) 14:166 DOI 10.1186/s12934-015-0346-x

TECHNICAL NOTES

WJWJWJ MICROBIAL CELL jjwjwj FACTORIES

Open Access

Cloning strategies for heterologous expression of the bacteriocin enterocin A by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475

CrossMark

Juan J. Jiménez1, Dzung B. Diep2, Juan Borrero1, Loreto Gútiez1, Sara Arbulu1, Ingolf F. Nes2, Carmen Herranz1, Luis M. Cintas1 and Pablo E. Hernández1*

Abstract

Background: Bacteriocins produced by lactic acid bacteria (LAB) attract considerable interest as natural and nontoxic food preservatives and as therapeutics whereas the bacteriocin-producing LAB are considered potential probiotics for food, human and veterinary applications, and in the animal production field. Within LAB the lactobacilli are increasingly used as starter cultures for food preservation and as probiotics. The lactobacilli are also natural inhabitants of the gastrointestinal (GI) tract and attractive vectors for delivery of therapeutic peptides and proteins, and for production of bioactive peptides. Research efforts for production of bacteriocins in heterologous hosts should be performed if the use of bacteriocins and the LAB bacteriocin-producers is ever to meet the high expectations deposited in these antimicrobial peptides. The recombinant production and functional expression of bacteriocins by lactobacilli would have an additive effect on their probiotic functionality.

Results: The heterologous production of the bacteriocin enterocin A (EntA) was evaluated in different Lactobacillus spp. after fusion of the versatile Sec-dependent signal peptide (SPusp4J) to mature EntA plus the EntA immunity gene (entA + entiA) (fragment UAI), and their cloning into plasmid vectors that permitted their inducible (pSIP409 and pSIP411) or constitutive (pMG36c) production. The amount, antimicrobial activity (AA) and specific antimicrobial activity (SAA) of the EntA produced by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475 transformed with the recombinant plasmids pSIP409UAI, pSIP411UAI and pMGUAI varied depending of the expression vector and the host strain. The Lb. casei CECT475 recombinant strains produced the largest amounts of EntA, with the highest AA and SAA. Supernatants from Lb. casei CECT (pSIP411UAI) showed a 4.9-fold higher production of EntA with a 22.8-fold higher AA and 4.7-fold higher SAA than those from Enterococcus faecium T136, the natural producer of EntA. Moreover, supernatants from Lb. casei CECT475 (pSIP411UAI) showed a 15.7- to 59.2-fold higher AA against Listeria spp. than those from E. faecium T136.

Conclusion: Lb. casei CECT457 (pSIP411UAI) may be considered a promising recombinant host and cell factory for the production and functional expression of the antilisterial bacteriocin EntA.

Keywords: Bacteriocins, Enterocin A, Lactic acid bacteria (LAB), Expression systems, Lactobacillus spp., Heterologous bacteriocin production

'Correspondence: ehernan@vet.ucm.es

1 Departamento de Nutrición, Bromatología y Tecnología de los

Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid

(UCM), Avenida Puerta de Hierro, s/n, 28040 Madrid, Spain

Full list of author information is available at the end of the article

O© 2015 Jiménez et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License Centfcll (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Background

Within lactic acid bacteria (LAB) the lactobacilli are increasingly used as starter cultures for food preservation and as probiotics [1]. The lactobacilli are also natural inhabitants of the gastrointestinal (GI) tract and attractive vectors for delivery of therapeutic peptides and proteins and production of bioactive peptides [2, 3]. Furthermore, most probiotics enhance intestinal barrier function, display immunomodulatory activity and exert protective effects against pathogens due to the production of antimicrobial compounds [4-6]. Since the in situ production of the antilisterial bacteriocin Abp118 is the major reason of the well-documented probi-otic effect of Lb. salivarius UCC118 against Listeria monocytogenes EFDe infections in mice [7, 8], the production of bacteriocins by lactobacilli surely would have an additive effect on their probiotic functionality.

Bacteriocins are ribosomally synthesized antimicrobial peptides secreted by bacteria, and those produced by LAB attract considerable interest as natural and nontoxic food preservatives, for human and veterinary applications, and in the animal production field [9, 10]. Most bacteriocins, including those produced by entero-cocci and named enterocins are synthesized as biologically inactive precursors or prepeptides containing an N-terminal extension of the so-called double-glycine type (leader sequence) that is cleaved concomitantly with export across the cytoplasmic membrane by dedicated ATP-binding cassette transporters (ABC transporters) and their accessory proteins [11]. However, many secreted prokaryotic proteins and a few bacteri-ocins contain N-terminal extensions of the Sec-dependent type (signal peptide) that are proteolytically cleaved concomitantly with peptide externalization by the general secretory pathway (GSP) or Sec-dependent pathway [12]. And the signal peptide (SP) of secretory proteins and bacteriocins may drive fused mature bacteriocins to SPs for their secretion by recombinant LAB [9, 10, 13]. The mature bacteriocins are often cationic, amphiphilic molecules of 20-60 amino acid residues that are classified into two main classes: the lantibiotics or class I that consist of modified bacteriocins and the class II or non-modified bacteriocins which are further subdivided in class IIa, class IIb, class IIc, and class IId. Among these subgroups, the class IIa bacteriocins (also referred to as pediocin-like bacteriocins) have attracted much attention due to their strong antilisterial activity [14]. Additional subgroups have been suggested for leaderless peptides, circular bacteriocins, linear peptides derived from large proteins, and the glycosylated bacteriocins [15].

Accordingly, bacteriocins with high antimicrobial activity against bacterial pathogens could be overproduced and would contribute to the probiotic effect of recombinant Lactobacillus spp. strains [8, 16]. Enterocin A (EntA)

is a class Ila bacteriocin whose synthesis is directed by the entAIFKRTD operon and from which entA encodes the enterocin A prepetide synthesized as an 18 amino acid leader sequence of the double-glycine type and the 47 amino acid mature bacteriocin [17, 18]. Moreover, its potent antilisterial activity has driven interest for its overproduction by LAB mostly of the genera Lactococcus, Ente-rococcus and Pediococcus [13, 19] and also by yeasts from the genera Pichia, Kluyveromyces, Hansenula and Arxula, throughout fusions of mature EntA to signal peptides (SPs) that act as secretion signals [20, 21]. Accordingly, of bio-technological interest would be the design and construction of recombinant Lactobacillus spp. for the controlled or constitutive heterologous production of bacteriocins with high antimicrobial activity against Listeria spp.

In this work, Lb. sakei Lb790 a non-bacteriocin producing strain from meat origin [22], Lb. plantarum NC8 from grass silage encoding the two-peptide plantaricins PlnEF, PlnJK and PLNC8a|3 of narrow inhibitory spectra [23, 24] and Lb. casei CECT475, a reported non-bacteriocin producer from dairy origin, were transformed with derivatives of the inducible protein expression vectors pSIP409 and pSIP411 and the constitutive pMG36c expression vector, for evaluation of the production of EntA and its functional expression as determined by evaluation of their antimicrobial activity against Listeria spp.

Results

Heterologous production and functional expression of EntA by different Lactobacillus spp. strains

Since the leader sequence of EntA (LSentA) is of a double-glycine type which restricts expression of the bacteriocin to limited LAB strains containing homologous dedicated ABC-transporters, we therefore employed the more versatile signal peptide SPusp45 for the Sec-dependent exter-nalization of mature EntA, as well as the use of protein expression vectors that permitted the inducible (pSIP409, pSIP411) or constitutive (pMG36c) production of the synthesized bacteriocin by different Lactobacillus spp. host strains. Thus, cloning of the lactococcal SPusp45 fused to mature entA (EntA) and entiA (EntI) (fragment UAI) into plasmids pSIP409, pSIP411 and pMG36c resulted in the plasmid derived vectors pSIP409UAI, pSIP411UAI and pMGUAI, respectively. Transformation of Lb. sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475 with plasmids pSIP409UAI, pSIP411UAI and pMGUAI yielded recombinant Lactobacillus spp.-derived strains which were further checked by bacteriocinogenicity tests, PCR and sequencing of the inserts. Halos of inhibition of variable sizes were observed by all transformed Lactobacillus spp. (results not shown), confirming that recombinant plasmids were responsible of their antimicrobial activity.

The production and functional expression of the EntA in supernatants of the recombinant Lactobacillus spp. strains was quantified using specific anti-EntA antibodies in a NCI-ELISA, and by a microtitre plate assay (MPA). None of the native Lactobacillus spp. strains showed production of EntA (Table 1). The production of EntA by Lb. sakei Lb790 (pSIP411UAI) and Lb. casei CECT475 (pSIP411UAI) was 2.7- and 4.9-fold higher, respectively, whereas production of EntA by Lb. plantarum NC8 (pSIP411UAI) was 4.7-times lower than production of EntA by the natural producer E. faecium T136. The production of EntA by Lb. sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475 transformed with either pSIP-409UAI or pMGUAI, was 1.1- to 6.3-times lower than production of EntA by E. faecium T136 (Table 1).

When supernatants of the recombinant Lb. sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475 strains were evaluated for their antimicrobial activity against E. faecium P13 (EntAS), the antimicrobial activity (AA) of Lb. sakei Lb790 (pSIP411UAI) was 2.2-fold higher while its specific antimicrobial activity (SAA) was 1.2-times lower than the EntA produced by E. faecium T136 (Table 1). Lb. sakei Lb790 (pSIP409UAI) showed 2.2-times lower AA and 1.5-times lower SAA and Lb. sakei Lb790 (pMGUAI) showed 15-times lower AA and

5.5-times lower SAA, when compared to the control EntA producer. All Lb. plantarum NC8 recombinants showed a 17.1- to 38-times lower AA and 3.6- to 6.0-times lower SAA, when compared to the control EntA producer. However, transformation of Lb. casei CECT475 with plasmids pSIP409UAI, pSIP411UAI and pMGUAI generated supernatants with 1.3-, 22.8- and 1.2-fold higher AAA and 4.3-, 4.7- and 2.1-fold higher SAA, respectively, than those from E. faecium T136 (Table 1).

Furthermore, the evaluation of the antimicrobial activity of supernatants from the recombinant Lb. sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475 against five Listeria spp. and six L. monocytogenes strains, showed that supernatants from Lb. sakei Lb790 (pSIP411UAI) displayed 3.8-times lower to 2.7-fold higher AA whereas those from Lb. sakei Lb790 (pSIP409UAI) and Lb. sakei Lb790 (pMGUAI) showed 1.8- to 9.1-times lower and a 6.0- to 45-times lower AA, respectively, than those from E. faecium T136. Supernatants from all recombinant Lb. plantarum NC8 strains showed a 4.9- to 120-times much lower AA than the control EntA producer (Table 2). However, despite the measurable and non-previously reported antimicrobial activity of Lb. casei CECT475, the supernatants from Lb. casei CECT475 (pSIP409UAI) showed 1.6- to 13.9-fold higher AA, those from Lb.

Table 1 Bacteriocin production and antimicrobial activity of supernatants from recombinant strains

Strain Bacteriocin production (pg/mg cell dry weight)3 Antimicrobial activity (BU/mg cell dry weight)b Specific antimicrobial activity (BU/pg EntA)c

Lactobacillus sakei

Lb790 NP NA NE

Lb790 (pSIP409UAI) 1.3 324 249

Lb790 (pSIP411UAI) 5.2 1578 303

Lb790 (pMGUAI) 0.7 48 68

Lactobacillus plantarum

NC8 NP NA NE

NC8 (pSIP409UAI) 0.4 42 105

NC8 (pSIP411UAI) 0.4 36 90

NC8 (pMGUAI) 0.3 19 63

Lactobacillus casei

CECT475 NP 102 NE

CECT475 (pSIP409UAI) 1.7 958 1629

CECT475 (pSIP411UAI) 9.3 16,466 1771

CECT475 (pMGUAI) 1.1 869 790

Enterococcus faecium

T136d 1.9 721 379

Most of the data are mean from two independent determinations in triplicate NP no production, NA no activity, NE not evaluable

a Production of EntA was calculated by using a NCI-ELISA with polyclonal antibodies specific for EntA b Antimicrobial activity was calculated against E. faecium P13 (EntAs). BU, bacteriocin units

c Specific antimicrobial activity refers to the antimicrobial activity against E. faecium P13 divided by the EntA produced d Culture of E. faecium T136 used as control for production and antimicrobial activity of EntA

Table 2 Antimicrobial activity of supernatants from recombinant Lactobacillus spp. strains against Listeria spp.a

Strain L ivanovii L grayi L. welshimeri L seeligeri L innocua L monocytogenes

CECT913 CECT931 CECT919 CECT917 CECT910 CECT911 CECT935 CECT936 CECT939 CECT4031 CECT4032

Lactobacillus sakei

Lb790 NA NA NA NA NA NA NA NA NA NA NA

Lb790 (pSIP409UAI) 1450 2575 1797 2012 1019 1387 1254 3317 508 545 1427

Lb790 (pSIP411 UAI) 5317 12,350 15,045 7866 6485 10,852 10,533 15,454 1661 1316 1418

Lb790 (pMGUAl) 897 643 537 297 191 361 264 701 584 598 407

Lactobacillus plantarum

NC8 NA NA NA NA NA NA NA NA NA NA NA

NC8 (pSIP409UAI) 641 551 255 360 140 288 250 623 451 853 224

NC8 (pSIP411 UAI) 920 960 484 137 462 653 721 1159 684 1010 162

NC8 (pMGUAl) 890 692 713 112 121 501 595 843 525 765 85

Lactobacillus casei

CECT475 965 920 103 793 201 243 307 524 506 356 890

CECT475 (pSIP409UAI) 44,433 37,711 21,713 21,916 24,091 36,639 42,977 57,145 48,839 59,408 18,740

CECT475 (pSIP411 UAI) 185,567 202,356 179,199 211,214 182,181 255,191 180,191 250,101 206,942 293,825 157,331

CECT475 (pMGUAl) 34,804 12,417 5619 7320 2656 4630 15,315 14,350 17,310 20,983 7379

Enterococcus faecium

T136b 9668 4599 5555 13,419 4582 4165 3876 5663 3495 4996 5096

Most of the data are mean from two independent determinations in triplicate NA no activity

a Antimicrobial activity expressed in BU per milligrams cell dry weight b Culture of E. faecium T136 used as control for antimicrobial activity of EntA

casei CECT475 (pSIP411UAI) showed 15.7- to 59.2-fold higher AA and those from Lb. casei CECT475 (pMGUAI) showed 0.54- to 4.9-fold higher AA than those from E. faecium T136 (Table 2).

Purification of EntA and mass spectrometry analysis

The EntA produced by Lb. sakei Lb790 (pSIP411UAI) and Lb. casei CECT475 (pSIP409UAI) was purified to homogeneity following a previously described chromatographic procedure (results not shown). MALDI-TOF MS analysis of the purified EntA from Lb. sakei Lb790 (pSIP411UAI) showed a major peptide fragment of a

molecular mass of 4842.62 Da (Fig. 1a), nearly identical to the EntA produced by different recombinant yeasts [20] while the purified EntA produced by Lb. casei CECT475 (pSIP411UAI) showed peptide fragments of different molecular massess among which a peptide fragment of 4844.53 Da, nearly identical to the observed molecular mass (4844.40 Da) of the EntA produced by E. faecium T136 [13], was also observed (Fig. 1b). In both purifications the peptide fragment of 4860.2 Da may correspond to oxidation (+16 Da) of the methionine residue (Met33) of the EntA to methionine sulfoxide (MetSO) (Fig. 1). The visualization by MALDI-TOF MS of peptide

Mass (m/z)

Mass (m/z)

Fig. 1 Mass spectrometry analysis of purified enterocin A from Lb. sakei Lb790 (pSIP411UAI) (a), and Lb. casei CECT475 (pSIP411UAI) (b)

fragments of different molecular massess (Fig. 1b) may suggest that the EntA produced by Lb. casei CECT475 (pSIP411UAI) has not been purified to homogeneity or that these peptides could be responsible of the low antimicrobial activity observed in supernatants of Lb. casei CECT475. However, treatment of crude supernatants of the control strain Lb. casei CECT474 with proteinase K (1 mg/ml) revealed that the antimicrobial activity of the supernatants was not of proteinaceous nature (results not shown).

Discussion

Lactobacilli are common colonisers of the human gastrointestinal and urogenital tracts, skin and the oral cavity and they merit recognition as starters in the production of fermented products, and as probiot-ics [25, 26]. They are also being evaluated for production of functional foods enriched in bioactive peptides [3]. Furthermore, production of bacteriocins by lac-tobacilli could find their use as natural antimicrobial peptides while the bacteriocin-producing lactobacilli could be evaluated for their improved functionality as probiotics. Several gene expression systems have been developed for efficient overproduction of heterologous proteins in LAB [1, 27, 28]. Previous studies have evaluated the production, secretion and functional expression of the EntA by different LAB, mostly of the genera Lactococcus, Enterococcus, and Pediococcus [13, 19, 29] and yeasts [20, 21]. However, of great biotechnologi-cal interest would be the construction of recombinant Lactobacillus spp. for production of bacteriocins with known and potent antimicrobial activity against Listeria spp.

For protein expression by Lb. sakei and Lb. plantarum but also for other Lactobacillus spp., the so-called pSIP expression vectors permits expression of the gene of interest under control of an inducible promoter by an externally added peptide pheromone [1, 28]. The pSIP system has been successfully applied for intracellular expression, secretion and surface anchoring of a variety of proteins in Lb. plantarum and Lb. sakei [1]. However, although these pSIP vectors have been evaluated for expression of different reporter proteins, they have not been yet fully evaluated for secretion and functional expression of bacteriocins. In these vectors the expression of genes of interest is driven by strong, regulated promoters derived from the bacteriocin sakacin P structural gene (PsppA) or the sakacin Q structural gene (PsppQ also recorded as PorfX) with an engineered NcoI site for translational fusion cloning, as well as for components of the cognate two-component signal transduction system (SppK and SppR) which responds to an externally added peptide pheromone (SppIP). These vectors also carries a

multicloning site (MCS) and the replicon derived from the narrow-host-range Lactobacillus replicon from plas-mid p256 (pSIP409) or the broad-host-range, high-copy-number replicon from plasmid pSH71 (pSIP411) [28]. The expression vector pMG36c contains the low copy replication origin of plasmid pWV01 and the strong P32 promoter to drive the constitutive transcription of inserted genes into the multicloning site (MCS) of pUC18 [30]. Different homologous and heterologous signal pep-tides (SPs) have been also evaluated for secretion of het-erologous proteins and bacteriocins by LAB, although expression yield and secretion efficiency are not only steered by the SP but also the host producer [1, 10, 13].

In this work, Lb. sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475 were transformed with the recombinant plasmids pSIP409UAI, pSIP411UAI and pMGUAI for heterologous production of EntA and evaluation of its functional expression against Listeria spp. The results obtained suggest that production, secretion and antimicrobial activity of the EntA produced depend on the expression vector and the host strain (Table 1). EntA producers are protected from the antagonistic effect of this bacteriocin by the concomitant expression of a cognate immunity protein (EntiA) and bacteriocins of the class IIa, such as the EntA use components of the mannose phosphotransferase system (Man-PTS) of the susceptible cells as the target/receptor. The immunity proteins form a strong complex with the receptor proteins, thereby preventing cells from being killed [15, 31]. Of interest is the 2.7- and 4.9-fold enhanced production of EntA by Lb. sakei Lb790 (pSIP411UAI) and Lb. casei CECT475 (pSIP-411UAI), respectively, as compared to the rest of recombinant Lactobacillus spp. and E. faecium T136 (Table 1). The production of EntA would depend, among other factors, on plasmid stability and copy number differences between pSIP409, pSIP411 and pMG36c but, more likely, might be caused by promoters used to drive gene expression. For optimization of protein production inducible systems are often considered superior to constitutive systems since the short induction time for bacteriocin production from the pSIP-inducible vectors most probably prevents EntA from attaching to cell walls, forming aggregates, and/or undergoing protease degradation [32]. The high-copy number replicon of pSIP411 may be also a contributing factor to the higher production of EntA by Lactobacillus spp. recombinants transformed with pSIP-411UAI instead of pSIP409UAI.

Protein secretion is a preferred means of protein expression in the development of LAB as cell factories for production of biologically active compounds [33]. However, it may happen that SPusp45 could modulate differently the secretion of EntA by the recombinant Lb. sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT47

hosts, as it appeared with secretion of EntA and other bacteriocins by different LAB [10, 13]. It may also happen that mature EntA remain N-terminally associated to the cell membrane of the producer cells via a Sec-type signal peptide that is not cleaved off during secretion [34]. The different molecular folding of EntA inside the less EntA-producing recombinant Lb. plantarum hosts may also maintain the prepeptide in an secretion-incompetent conformation [35]. It is known that Lb. plantarum NC8 encodes three two-peptide plantaricins of narrow inhibitory spectra, regulated by a quorum sensing based network, but unable to produce bacteriocins as pure cultures in liquid media [24]. Thus, variations in bacteriocin secretion capacities may be also governed by autoinducer peptide production and recognition and post-transcrip-tional factors such as codon usage, mRNA stability and translational efficiency that may steer EntA production from the recombinant Lb. sakei Lb790 and Lb. plantarum NC8 [36]. New variants of the modular pSIP-vectors, encoding different SPs, have been tested for inducible gene expression and reporter protein secretion in Lacto-bacillus spp. All recombinant strains secreted the target protein nuclease A (NucA), albeit with different production levels [1].

In this work, polyclonal antibodies of predetermined specificity for EntA and an NCI-ELISA have permitted evaluation of the specific antimicrobial activity (SAA) of the produced EntA against E. faecium P13 (EntAS). From the Lb. sakei Lb790-derived recombinants, only Lb. sakei Lb790 (pSIP411UAI) showed a 2.2-fold higher antimicrobial activity (AA) but a 1.2-times lower SAA than the EntA produced by E. faecium T136 (Table 1). All Lb. plantarum NC8 recombinants showed a much lower AA and SAA when compared to the control EntA producer. However, all Lb. casei CECT475-derived recombinants generated supernatants with higher AA and SAA than those from E. faecium T136. Of interest is the 22.8-fold higher AA and the 4.7-fold higher SAA of supernatants of Lb. casei CECT475 (pSIP411UAI) (Table 1). According to these results, it is important to consider that not always a higher bacteriocin production by recombinant LAB may report a higher AA and SAA [9, 10]. The low AA and SAA of the EntA produced by the Lb. sakei Lb790- and Lb. plantarum NC8-hosts may depend on many factors which are difficult to determine. It is possible that: (1) regulatory responses to secretion stress activate quality control networks of the producer cells involving folding factors and housekeeping proteases [37], (2) differences in the Sec-dependent translocation and Sec-machinery, differences in protein folding, and conformational modifications of bacteriocins to a less extracellular active form may also decrease the antagonistic activity of the secreted EntA [38], (3) secretion of

truncated bacteriocins may also lower the antimicrobial activity of the producer cells [10], (4) the formation of disulfide bonds (DSB) from the four cysteine residues in EntA may also play a role in the folding, structural integrity, and antimicrobial activity of the produced bacteriocin [39], and (5) the EntA contains a methio-nine residue that may change to an apparently less active form due to its oxidation to methionine sulfoxide [40]. The lower AA and SAA of the produced EntA may be also adscribed to differences in protein folding efficiency and bacteriocin self-aggregation [13]. Although Lb. sakei and Lb. plantarum have been considered appropriate hosts for the recombinant production of a number of reporter proteins and enzymes [1, 41-43], the results of this work resolve Lb. casei CECT475 as the preferred host for heterologous production and functional expression of the bacteriocin EntA.

Supernatants from all recombinant Lb. casei CECT475 hosts, producers of EntA, showed up to a 59.2-fold higher AA against Listeria spp. than any other Lb. sakei Lb790-or Lb. plantarum NC8-recombinant producer of EntA (Table 2). Furthermore, Lb casei CECT (pSIP411UAI) an inducible overproducer of EntA with higher AA and SAA in its supernatants than those from E. faecium T136, could be considered as a cellular factory and an alternative to E. faecium T136 for production and recovery of the highly active antilisterial bacteriocin EntA. The controlled production of EntA by Lb. casei CECT475 (pSIP411UAI) and the constitutive production of this bacteriocin by Lb. casei CECT475 (pMGUAI) could be also evaluated as a contributing antilisterial effect of Lb. casei CECT475, also cited as Lb. casei ATCC393, during further evaluation of the potential of the Lb. casei CECT475-derived recombinant strains during production of dry-fermented sausages [44, 45], production of antithrombotic and angiotensin converting enzyme (ACE)-inhibitory peptides (ACEIP) from bovine casein [46] or during production of antioxidant and antimuta-genic peptides from yogurt [3].

Conclusions

The use of Lb. casei CECT475-derived strains, generally recognized as safe (GRAS) and with a qualified presumption of safety (QPS), as recombinant bacteriocin producers may provide means by which the potential benefits of antimicrobial compounds can be exploited in the food industry, human and veterinary applications, and in the animal production field. The combined use of the induc-ible protein expression vector pSIP411 and Lb. casei CECT475 as the producer host, would also merit recognition as a novel gene expression system for the efficient overproduction and functional expression of EntA by Lb. casei.

Methods

Microbial strains, plasmids, and growth conditions

The microbial strains and plasmids used in this study are listed in Table 3. Enterococcus faecium T136 was used as the source of entA (EntA) and entiA (EntI), whereas Lactococcus lactis MG1363 was the source of the signal peptide from protein Usp45 (SPusp45). The lactococcal strains were propagated at 32 °C in M17 broth (Oxoid Ltd., Basingstoke, UK) supplemented with 0.5 % (w/v) glucose (GM17). The enterococcal strains and the lac-tobacilli were grown in MRS broth (Oxoid) at 32 °C. Escherichia coli XL10 Gold (Stratagene, La Jolla, CA, USA) was grown in BHI (Oxoid) broth at 37 °C with shaking. Listeria spp. strains were cultured in BHI broth (Oxoid) at 32 °C. Agar plates were made by addition of 1.5 % (w/v) agar (Oxoid) to the liquid media. When necessary, chloramphenicol (Sigma-Aldrich Inc., St. Louis, MO, USA) was added at 10 |g ml-1 for E. coli, lactococci

and lactobacilli. Erythromicin (Sigma) was added at 350 |g ml-1 for E. coli and at 10 |g ml-1 for lactococci and lactobacilli. Cell dry weights of late exponential phase cultures expressed as cell dry mass were determined gravimetrically.

Basic genetic techniques and enzymes

Total genomic DNA from L. lactis MG1363 and E. faecium T136 was isolated using the Wizard® DNA Purification Kit (Promega, Madison, WI, USA). Plasmid DNA isolation was carried out using the QIAprep Spin Mini-prep Kit (QIAGEN, Hilden, Germany), as suggested by the manufacturer, but cells were suspended with lysozyme (40 mg ml-1) and mutanolysin (500 U ml-1) and incubated at 37 °C for 10 min before following the kit instructions. DNA restriction enzymes were supplied by New England Biolabs (Beverly, MA, USA). Ligation reactions were performed with the T4 DNA ligase (Roche Molecular

Table 3 Bacterial strains and plasmids used in this study

Strain or plasmid Description1 Source and/or

referenceb

Strains

Lactobacillussakei Lb790 Host strain, meat isolate, non-bacteriocin producer [22]

Lactobacillusplantarum NC8 Host strain, silage isolate, plasmid free [48]

Lactobacillus casei CECT475 Host strain, cheese isolate, also recorded as strain ATCC393 CECT

Lactococcus lactis MG1363 Source of SPusp45, plasmid-free and prophage-cured derivative of L. lactis NCDO 712 [51]

Enterococcus faecium T136 Enterocin A and B producer, source of entA and entiA, control strain DNBTA [52]

Enterococcus faecium P13 Enterocin P producer, control strain MPA and ADT indicator DNBTA [52]

Listeria ivanovii CECT913 Indicator strain, sheep isolate CECT

Listeria grayi CECT931 Indicator strain, chinchilla faeces CECT

Listeria welshimeri CECT919 Indicator strain, decaying vegetation CECT

Listeria seeligeri CECT917 Indicator strain, soil isolate CECT

Listeria innocua CECT910 Indicator strain, cow brain isolate CECT

Listeria monocytogenes CECT911 Indicator strain, spinal fluid of man CECT

Listeria monocytogenes CECT935 Indicator strain, spinal fluid of child CECT

Listeria monocytogenes CECT936 Indicator strain, origin not described CECT

Listeria monocytogenes CECT939 Indicator strain, chicken isolate CECT

Listeria monocytogenes CECT4031 Indicator strain, rabbit isolate CECT

Listeria monocytogenes CECT4032 Indicator strain, soft cheese isolate CECT Plasmids

pSIP409 Emr; pSIP401 with 256rep and PorfX::gusA [28]

pSIP411 Emr; pSIP401 with SH71 rep and PorfX::gusA [28]

pMG36c Cmr, pMG36e derivative RUG-MG [30]

pSIP409UAI Emr; pSIP409 derivative encoding the PCR product UAI (SPusp45 fused to mature entA and This work

entiA genes)

pSIP411UAI Emr; pSIP411 derivative encoding the PCR product UAI (SPusp45 fused to mature entA and This work

entiA genes)

pMGUAI Cmr, pMG36c derivative encoding the SPusp45 fused to mature entA and entiA genes) [13] a ADT, agar well diffusion test; MPA, microtitre plate asay; Cmr, chloramphenicol resistance; Emr, erythromycin

b CECT, Colección Española de Cultivos Tipo (Valencia, Spain); DNBTA, Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid (Madrid, Spain); RUG-MG, Department of Molecular Genetics, University of Groningen (Haren, The Netherlands)

Biochemicals, Mannheim, Germany). E. coli XL10 Gold competent cells were transformed as described by the supplier (Stratagene). Competent L. lactis MG363 and Lactobacillus spp. cells were electrotransformed with a Gene Pulser™ and Pulse Controller apparatus (Bio-Rad Laboratories, Hercules, CA, USA), according to Holo and Nes [47] and Aukrust and Blom [48], respectively.

PCR amplification and nucleotide sequencing

Oligonucleotide primers were obtained from Sigma-Genosys Ltd. (Cambridge, UK). PCR-amplification of inserts was performed as previously described [13]. The PCR-generated fragments were purified by a Nucle-oSpin® Extract II Kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) for cloning and nucleotide sequencing. Nucleotide sequencing of purified PCR products was done using the ABI PRISM® BigDye™ Terminator cycle sequence reaction kit and the automatic DNA sequencer ABI PRISM, model 377 (Applied Biosystems, Foster City, CA, USA), at the Unidad de Genómica (Facultad de Ciencias Biológicas, Universidad Complutense de Madrid, Madrid, Spain).

Recombinant plasmids derived from pSIP409, pSIP411 and pMG36c

The primers and inserts used for the construction of the recombinant plasmids derived from pSIP409 and pSIP411 are listed in Table 4. Plasmid derivatives were constructed as follows: the primer pair USPNC-F/JJ8-R was used for PCR-amplification from total genomic DNA of L. lactis MG1363 of a 124-pb NcoI fragment (UA) encoding the SPusp45, with a tail complementary to the DNA encoding the N-terminal sequence of EntA. Primers JJ3-F/JJ5-R were used for PCR-amplification from total genomic DNA of E. faecium T136 of a 475-bp XhoI fragment (AI) containing mature entA and entiA. Mixtures of fragments UA and AI were used as templates to amplify the 567-bp NcoI/XhoI fragment UAI encoding the mature entA and entiA fused to the SPusp45. Fragment UAI was digested

Table 4 Primers and PCR products used in this study

Primer or PCR Nucleotide sequence (5'-3') or description

product

Primers JJ3-F JJ5-R USPNC-F JJ8-R PCR products AI UA UAI

with the corresponding restriction enzymes and inserted into either pSIP409 and pSIP411, digested with NcoI/XhoI. The ligation mixtures were used to transform E. coli XL10 Gold and L. lactis MG1363 competent cells, respectively, and the selected plasmid derivatives pSIP409UAI and pSIP411UAI were checked by bacteriogenicity tests, PCR and sequencing of the inserts. The construction of plas-mid pMGUAI has been described previously [13]. Plasmids pSIP409UAI, pSIP411UAI and pMGUAI were used to transform competent cells of Lb. sakei Lb790, Lb. plan-tarum NC8 and Lb. casei CECT475.

Antimicrobial activity of the recombinant Lactobacillus spp. strains

The antimicrobial activity of colonies from the recombinant Lactobacillus spp. strains was examined by the stab-on-agar test (SOAT), as previously described [49]. When appropriate, cultures were induced with 50 ng ml-1 of the inducing peptide SppIP [50] at an OD600 of, approximately, 0.3 and the induced cultures were grown at 30 °C for 5 h. Cell-free culture supernatants were obtained by centrifugation of cultures at 12,000 xg at 4 °C for 10 min, adjusted to pH 6.2 with 1 M NaOH, filtered through 0.2 |im pore-size filters (Whatman Int. Ltd., Maidstone, UK), and stored at -20 °C until use. The antimicrobial activity of the supernatants was quantified by a microti-ter plate assay (MPA), as previously described [13], using E. faecium P13 as the indicator microorganism. With the MPA, growth inhibition of the sensitive culture was measured spectrophotometrically at 620 nm with a microtitre Labsystems iEMS plate reader (Labsystems, Helsinki, Finland). One bacteriocin unit (BU) was defined as the reciprocal of the highest dilution of the bacteriocin causing 50 % growth inhibition (50 % of the turbidity of the control culture without bacteriocin). The antimicrobial activity of the recombinant Lactobacillus spp. hosts was also tested against selected Listeria spp. obtained from the CECT (Colección Española de Cultivos Tipo, Valencia, Spain), using the MPA.

Amplification

ACCACTCATAGTGGAAAATATTATGG AI

GGCGGAGCTCTCCAGGCATTAAAATTGAGATTTATCTCCATAATC AI, UA, UAI

GAATTCTCACCATGGGAAAAAAAAAGATTATCTCAGCTATTTTAATGTCTAC UA, UAI

CCATAATATTTTCCACTATGAGTGGTAGCGTAAACACCTGACAACGG UA

475-bp XhoI fragment containing the mature enterocin A (entA) and immunity (entiA) genes 124-pb NcoI fragment containing the usp45 signal peptide (SPusp45) and the begining of mature entA 567-bp Nco/XhoI fragment containing the SPusp45 fused to mature entA and entiA

ELISA for detection and quantification of EntA

Polyclonal antibodies with predetermined specificity for EntA and a non-competitive indirect enzyme-linked inmunosorbent assay (NCI-ELISA) were used to detect and quantify EntA in supernatants of the recombinant Lactobacillus spp. strains, essentially as described [13]. Briefly, wells of flat-bottom polystyrene microtitre plates (Maxisorp, Nunc, Roskilde, Denmark) were coated overnight (4 °C) with supernatants from E. faecium T136 or the recombinant strains. After addition of the anti-EntA specific antibodies and the goat anti-rabbit immunoglobulin G peroxidase conjugate (Cappel Laboratories, West Chester, PA, USA), bound peroxidase was determined with ABTS (2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid]) (Sigma) as the substrate by measuring the absorbance of the wells at 405 nm with a Labsystems iEMS reader (Labsystems) with a built-in software package for data analysis.

Purification of EntA and mass spectrometry analyses

EntA was purified from Lb. sakei Lb790 (pSIP411UAI) and Lb. casei CECT475 (pSIP411UAI), as previously described [13]. Briefly, supernatants from early stationary phase 1-L cultures of the recombinant Lactobacillus spp. strains were precipitated with ammonium sulfate, desalted by gel filtration, and subjected to cation-exchange and hydrophobic-interaction chromatography, followed by reverse-phase chromatography in a fastprotein liquid chromatography system (RP-FPLC) (GE Healthcare, Barcelona, Spain). Purified fractions were subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as previously described [13].

Authors' contributions

JJJ carried out the cloning experiments, the immunoassays and the purification of the bacteriocin enterocin A (EntA), participated in the design of the experiments and drafted the manuscript. JB, LG and SA participated in the cloning and transforming experiments, prepared competent cells and worked in the obtention of the anti-EntA rabbit polyclonal antibodies and design of the immunoassays. DBD, IFN, CH, LMC and PEH participated in the coordination and design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.

Author details

1 Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid (UCM), Avenida Puerta de Hierro, s/n, 28040 Madrid, Spain. 2 Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, 1432 As, Norway.

Acknowledgements

The authors express their gratitude to Prof. L. Axelsson (NOFIMA, The Norwegian Institute of Food, Fisheries and Aquaculture Research) and Prof. J. Kok (Department of Genetics, University of Groningen, The Netherlands), for supplying plasmids pSIP409 and pSIP411, and pMG36c, respectively. This work was partially supported by Grants AGL2012-34829 from the Ministerio de Economía y Competitividad (MINECO) and AGL2009-08348 from the Ministerio de Ciencia e Innovación (MICINN), by Grant GR35-10A from the BSCH-UCM, and by Grant S2013/ABI-2747 from the Comunidad de Madrid (CAM). J. J. Jiménez was recipient of a fellowship (FPI) from the Ministerio de Ciencia e

Innovación (MICINN), J. Borrero held a research contract from the CAM, L. Gútiez held a fellowship (FPU) from the Ministerio de Educación y Ciencia (MEC), and S. Arbulu held a fellowship (FPI) from the Ministerio de Economía y Competitividad (MINECO), Spain.

Competing interests

The authors declare that they have no competing interests.

Received: 24 August 2015 Accepted: 23 September 2015 Published online: 15 October 2015

References

1. Karlskas IL, Maudal K, Axelsson L, Rud I, Eijsink VGH, Mathiesen G. Heterologous protein secretion in lactobacilli with modified pSIP vectors. PLoS One. 2014;9(Suppl 3):e91125.

2. Hazerbrouck S, Oozeer R, Adel-Patient K, Langella P, Rabot S, Wal JM, Cor-thier G. Constitutive delivery of bovine ß-lactoglobulin to the digestive tracts of gnotobiotic mice by engineered Lactobacillus casei. Appl Environ Microbiol. 2006;72:7460-7.

3. Sah BNP, Vasiljevic T, McKechnie S, Donkor ON. Effect of probiotics on antioxidant and antimutagenic activities of crude peptide extract from yoghourt. Food Chem. 2014;156:264-70.

4. Corr SC, Hill C, Gahan CGM. Understanding the mechanisms by which probiotics inhibit gastrointestinal pathogens. Adv Food Nutr Res. 2009;56:1-15.

5. Riboulet-Bisson E, Sturme MHJ, Jeffery IB, O'Donnell MM, Neville BA, Forde BM, Claesson MJ, Harris H, Gardiner GE, Casey PG, Lawlor PG, O'Toole PW, Ross RP. Effect of Lactobacillussalivarius bacteriocin Abp118 on the mouse and pig intestinal microbiota. PLoS One. 2012;7(Suppl 2):e31113.

6. van Hemert S, Meijerink M, Molenaar D, Bron PA, de Vos P, Kleerebezem M, Wells JM, Marco ML. Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells. BMC Microbiol. 2010;10:293.

7. Corr SD, Li Y, Riedel CU, O'Toole PW, Hill C, Gahan GGM. Bacteriocin production as a mechanism for the antiinfective activity of Lactobacillus salivarius UCC118. Proc Natl Acad Sci USA. 2007;104:7617-21.

8. Dobson A, Cotter PD, Ross RP, Hill C. Bacteriocin production: a probiotic trait? Appl Environ Microbiol. 2012;78:1-6.

9. Borrero J, Jiménez JJ, Gútiez L, Herranz C, Cintas LM, Hernández PE. Use of the usp45 lactococcal secretion sequence signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis. Appl Microbiol Biotechnol. 2011;89:131-43.

10. Jiménez JJ, Borrero J, Diep DB, Gútiez L, Nes IF, Herranz C, Cintas LM, Hernández PE. Cloning, production and functional expression of the bacteriocin sakacin A (SakA) and two SakA-derived chimeras in lactic acid bacteria (LAB) and the yeasts Pichia pastoris and Kluyveromyces lactis. J Ind Microbiol Biotechnol. 2013;40:977-93.

11. Havarstein LS, Diep DB, Nes IF. A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitantly with export. Mol Microbiol. 1995;16:229-40.

12. Natale P, Brüsser T, Driessen AJM. Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane: distinct translocases and mechanisms. Biochim Biophys Acta. 2008;1778:1735-56.

13. Borrero J, Jiménez JJ, Gútiez L, Herranz C, Cintas LM, Hernández PE. Protein expression vector and secretion signal peptide optimization to drive the production, secretion, and functional expression of the bacteriocin enterocin A in lactic acid bacteria. J Biotechnol. 2011;156:76-86.

14. Nes IF, Yoon SS, Diep DB. Ribosomally synthesized antimicrobial peptides (bacteriocins) in lactic acid bacteria: a review. Food Sci Biotechnol. 2007;16:675-90.

15. Kjos M, Borrero J, Opsata M, Birri DJ, Holo H, Cintas LM, Snipen L, Hernández PE, Nes IF, Diep DB. Target recognition, resistance, inmunity and genome mining of class II bacteriocins from Gram-positive bacteria. Microbiology. 2011;157:3256-67.

16. Cotter PD, Ross RP, Hill C. Bacteriocins—a viable alternative to antibiotics? Nat Microbiol Rev. 2013;11:95-105.

17. Nilsen T, Nes IF, Holo H. An exporter inducer regulates bacteriocin pro- 38. duction in Enterococcus faecium CTC492. J Bacteriol. 2007;180:1848-54.

18. O'Keefe T, Hill C, Ross RR Characterization and heterologous expression of

the genes encoding enterocin A production, immunity and regulation in 39. Enterococcus faecium DRC1146. Appl Environ Microbiol. 1999;65:1506-15.

19. Martín M, Gutiérrez J, Criado R, Herranz C, Cintas LM, Hernández RE. Cloning, production and expression of the bacteriocin enterocin A produced

by Enterococcus faecium RLBC21 in Lactococcus lactis. Appl Microbiol 40.

Biotechnol. 2007;76:667-75.

20. Borrero J, Kunze G, Jiménez JJ, Böer E, Gútiez L, Herranz C, Cintas LM, Hernández RE. Cloning, production and functional expression of the 41. bacteriocin enterocin A, produced by Enterococcus faecium T136, by the

yeasts Pichiapastoris, Kluyveromyces lactis, Hansenulapolymorpha and

Arxula adeninivorans. Appl Environ Microbiol. 2012;78:5956-61. 42.

21. Jiménez JJ, Borrero J, Gútiez L, Arbulu S, Herranz C, Cintas LM, Hernández RE. Use of synthetic genes for cloning, production and functional expression of the bacteriocins enterocin A and bacteriocin E 50-52 by Pichia

pastoris and Kluyveromyces lactis. Mol Biotechnol. 2014;56:571-83. 43.

22. Schillinger U, Lücke FK. Antibacterial activity of Lactobacillus sake isolated from meat. Appl Environ Microbiol. 1989;55:1901-6.

23. Diep DB, Straume D, Kjos M, Torres C, Nes IF. An overview of the

mosaic bacteriocin pln loci from Lactobacillusplantarum. Reptides. 44.

2009;30:1562-74.

24. Maldonado-Barragán A, Ruíz-Barba JL, Jiménez-Díaz R Knockout of three component regulatory systems reveal that the apparently constitutive plantaricin- 45. production phenotype shown by Lactobacillus plantarum on solid medium is regulated via quorum sensing. Int J Food Microbiol. 2009;130:35-42.

25. Lebeer S, Vanderleyden J, De Keersmacker SCJ. Genes and molecules 46. of lactobacilli supporting probiotic action. Microbiol Mol Biol Rev. 2008;72:728-64.

26. Tsapieva A, Duplik N, Suvorov A. Structure of plantaricin locus of Lactobacillus plantarum 8R-A3. Benef Microb. 2011;2:255-61.

27. Mierau I, Kleerebezem M. 10 years of the nisin-controlled gene expres- 47. sion system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol. 2005;68:705-17.

28. Sorvig E, Mathiesen G, Naterstad K, Eijsink VGH, Axelsson L. High- 48. level, inducible gene expression in Lactobacillus sakei and Lactoba-

cillus plantarum using versatile expression vectors. Microbiology. 49.

2005;151:2439-49.

29. Martínez JM, Kok J, Sanders JW, Hernández RE. Heterologous co-production of enterocin A and pediocin RA-1 by Lactococcus lactis: detection by specific peptide-directed antibodies. Appl Environ Microbiol. 50.

2000;66:3543-9.

30. van de Guchte M, van der Vossen JMBM, Kok J, Vemena G. Construction

of a lactococcal expression vector: expression of hen egg white lysozyme 51. in Lactococcus lactis subsp lactis. Appl Environ Microbiol. 1989;55:224-8.

31. Diep DB, Skaugen M, Salehian Z, Holo H, Nes IF. Common mechanisms

of target cell recognition and immunity for class II bacterocins. Rroc Natl 52. Acad Sci USA. 2007;104:2384-9.

32. Gutiérrez J, Larsen R, Cintas LM, Kok J, Hernández RE. High-level heterologous production and functional expression of the sec-dependent enterocin R from Enterococcus faecium R13 in Lactococcus lactis. Appl Microbiol Biotechnol. 2006;72:41-51.

33. Mathiesen G, Sveen A, Riard JC, Axelsson L, Eijsink VGH. Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides. J Appl Microbiol. 2008;105:215-26.

34. Böhle LA, Riaz T, Egge-Jacobsen W, Skaugen M, Busk ÖL, Eijsink VGH, Mathiesen G. Identification of surface proteins in Enterococcus faecalis V583. BMC Genom. 2011;12:135.

35. Mathiesen G, Sveen A, Brurberg MB, Fedriksen L, Axelsson L, Eijsink VGH. Genome-wide analysis of signal peptide funtionality in Lactobacillus plantarum WCFS1. BMC Genom. 2009;10:425.

36. Diep DB, Mathiesen G, Eijsink VGH, Nes IF. Use of lactobacilli and their pheromone-based regulatory mechanism in gene expression and drug delivery. Curr Rharm Biotechnol. 2009;10:62-73.

37. Darmon E, Noone D, Masson A, Bron S, Kuipers OR, Devine KM, van Dijl JM. A novel class of heat and secretion stress-responsive genes is controlled by the autoregulated CssRS two-component system of Bacillus subtilis. J Bacteriol. 2002;184:5661-71.

Sarvas M, Harwood CR, Bron S, van Dijl JM. Rost-translocational folding of secretory proteins in Gram-positive bacteria. Biochim Biophys Acta. 2004;1694:311-27.

Freitas DA, Leclerc S, Miyoshi A, Oliveira SC, Sommer RSM, Rodrigues L, Correa A, Gautier M, Langella R, Azevedo VA, Le Loir Y. Secretion of Strep-tomyces tendae antifungal protein 1 by Lactococcus lactis. Braz J Med Biol Res. 2005;38:1585-92.

Johnsen L, Fimland G, Eijsink V, Nissen-Meyer J. Engineering increased stability in the antimicrobial peptide pediocin RA-1. Appl Environ Microbiol. 2000;66:4798-802.

Böhmer N, Lutz-Whal S, Fisher L. Recombinant production of hyper-thermostable CelB from Pyrococcus furiosus in Lactobacillus spp. Appl Microbiol Biotechnol. 2012;96:903-12.

Halbmayr E, Mathisen G, Nguyen TH, Maischberger T, Reterbauer CK, Eijsink VGH, Haltrich D. High-level expression of recombinant ß-galactosidases in Lactobacillus plantarum and Lactobacillus sakei using a sakacin R-based expression system. J Agric Food Chem. 2008;56:4710-9. Moraís S, Shterzer N, Grinberg IR, Mathiesen G, Eijsink VGH, Axelsson L, Lamed R, Bayer EA, Mizrahi I. Establishment of a Lactobacillus plantarum cell consortium for cellulase-xylanase sinergystic interactions. Appl Environ Microbiol. 2013;79:5242-9.

Sayas-Barberá E, Viuda-Martos M, Fernández-López F, Rérez-Alvarez JA, Sendra E. Combined use of a probiotic culture and citrus fiber in a traditional sausage "Longaniza de Rascua". Food Cont. 2012;27:343-50. Sidira M, Galanis A, Nikolau A, Kanellaki M. Evaluation of Lactobacillus casei ATCC 393 protective effect against spoilage of probiotic dry-fermented sausages. Food Cont. 2014;42:315-30.

Rojas-Ronquillo R, Cruz-Guerrero A, Flores-Nájera A, Rodríguez-Serrano G, Gómez-Ruíz L, Reyes-Grajeda JR, Jiménez-Guzmán J, García-Garibay M. Antithrombotic and angiotensin-converting enzyme inhibitory properties of peptides released from bovine casein by Lactobacillus casei Shirota. Int Dairy J. 2012;26:147-54.

Holo H, Nes IF. High-frequency transformation by electroporation of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol. 1989;55:3119-23. Aukrust T, Blom H. Transformation of Lactobacillus strains used in meat and vegetable fermentations. Food Res Int. 1992;25:253-61. Cintas LM, Casaus R, Havarstein LS, Hernández RE, Nes IF. Biochemical and genetic characterization of enterocin R, a novel sec-dependent bacteriocin from Enterococcus faecium R13 with a broad antimicrobial spectrum. Appl Environ Microbiol. 1997;63:4321-30.

Eijsink VGH, Brurberg M. Hans Middelhoven R, Nes IF. Induction of bacteriocin production in Lactobacillus sake by a secreted peptide. J Bacteriol. 1996;178:2232-43.

Gasson MJ. Rlasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococcci after protoplast-induced curing. J Bacteriol. 1983;154:1-9.

Casaus R, Nilsen T, Cintas LM, Nes IF, Hernández RE, Holo H. Enterocin B, a new bacteriocin from Enterococcus faeciumT136 which can act synergis-tically with enterocin A. Microbiology. 1997;143:2287-94.

Submit your next manuscript to BioMed Central and take full advantage of:

• Convenient online submission

• Thorough peer review

• No space constraints or color figure charges

• Immediate publication on acceptance

• Inclusion in PubMed, CAS, Scopus and Google Scholar

• Research which is freely available for redistribution

Submit your manuscript at www.blomedcentral.com/submlt

Central