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Article

Design of Lactococcus lactis Strains Producing Garvicin A and/or Garvicin Q, Either Alone or Together with Nisin A or Nisin Z and High Antimicrobial Activity against Lactococcus garvieae

by
Javier Feito
1,
Carlos Araújo
1,
Sara Arbulu
1,
Diogo Contente
1,
Beatriz Gómez-Sala
2,3,
Lara Díaz-Formoso
1,
Estefanía Muñoz-Atienza
1,
Juan Borrero
1,
Luis M. Cintas
1 and
Pablo E. Hernández
1,*
1
Grupo de Seguridad y Calidad de los Alimentos por Bacterias Lácticas, Bacteriocinas y Probióticos (Grupo SEGABALBP), Sección Departamental de Nutrición y Ciencia de los Alimentos (Higiene y Seguridad Alimentaria), Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro, s/n., 28040 Madrid, Spain
2
APC Microbiome Ireland, University College Cork, T12 K8AF Cork, Ireland
3
Teagasc Food Research Centre, Moorepark, P61 C996 Cork, Ireland
*
Author to whom correspondence should be addressed.
Foods 2023, 12(5), 1063; https://doi.org/10.3390/foods12051063
Submission received: 2 January 2023 / Revised: 20 February 2023 / Accepted: 24 February 2023 / Published: 2 March 2023
(This article belongs to the Special Issue Recent Advances in Lactic Acid Bacteria and Metabolite for Food Quality)
Browse Figure
Versions Notes

Abstract

:
Lactococcus garvieae is a main ichthyopathogen in rainbow trout (Oncorhynchus mykiss, Walbaum) farming, although bacteriocinogenic L. garvieae with antimicrobial activity against virulent strains of this species have also been identified. Some of the bacteriocins characterized, such as garvicin A (GarA) and garvicin Q (GarQ), may show potential for the control of the virulent L. garvieae in food, feed and other biotechnological applications. In this study, we report on the design of Lactococcus lactis strains that produce the bacteriocins GarA and/or GarQ, either alone or together with nisin A (NisA) or nisin Z (NisZ). Synthetic genes encoding the signal peptide of the lactococcal protein Usp45 (SPusp45), fused to mature GarA (lgnA) and/or mature GarQ (garQ) and their associated immunity genes (lgnI and garI, respectively), were cloned into the protein expression vectors pMG36c, which contains the P32 constitutive promoter, and pNZ8048c, which contains the inducible PnisA promoter. The transformation of recombinant vectors into lactococcal cells allowed for the production of GarA and/or GarQ by L. lactis subsp. cremoris NZ9000 and their co-production with NisA by Lactococcus lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24. The strains L. lactis subsp. cremoris WA2-67 (pJFQI), a producer of GarQ and NisZ, and L. lactis subsp. cremoris WA2-67 (pJFQIAI), a producer of GarA, GarQ and NisZ, demonstrated the highest antimicrobial activity (5.1- to 10.7-fold and 17.3- to 68.2-fold, respectively) against virulent L. garvieae strains.

1. Introduction

Bacteriocins produced by lactic acid bacteria (LAB) have been largely valued as potential food preservatives, and the LAB producers of bacteriocins (bacteriocinogenic strains) have been valued as potential starter, protective, probiotic, paraprobiotic and postbiotic cultures [1,2,3]. Moreover, concerns regarding the increase in antimicrobial resistances (AMRs) confer bacteriocins and the bacteriocinogenic LAB unlimited possibilities for applications in the food industry, human and veterinary medicine and the animal production field [4,5,6]. Microbial-derived biotics, including bacteriocins, are recognized as functional components of natural and bioengineered probiotic, paraprobiotic and postbiotic cultures [2,7,8]. Bacteriocins, including nisin A (NisA) and nisin Z (NisZ), drive the apoptosis of cancer cells and show low toxicity toward normal cells, making them promising anticancer candidates to replace or be combined with conventional therapeutic agents [9,10,11]. Bacteriocinogenic LAB and the bacteriocins they produce may also have an impact in the modulation of the microbiota and immune system of their host [12,13].
Most LAB bacteriocins are synthesized as biologically inactive precursors that contain an N-terminal extension, whereas the mature peptides are often cationic, amphiphilic, membrane-permeabilizing molecules. The N-terminal extensions of most bacteriocins are of the so-called double-glycine type (leader sequence) and are cleaved off concomitantly with export across the cytoplasmic membrane by dedicated, ATP-binding cassette transporters (ABC-transporters) and their accessory proteins [14]. However, a few bacteriocins contain N-terminal extensions of the Sec-type (signal peptide), which are proteolytically cleaved concomitantly with peptide externalization by the general secretory pathway (GSP) or the Sec-dependent pathway [15]. Several bacteriocins have been shown to be synthesized without N-terminal sequences and represent bacteriocins with a novel secretion mechanism [16].
The mature bacteriocins are often classified into three main classes: class I, or lantibiotics, which have post-translationally modified amino acid residues, class II bacteriocins, which have unmodified amino acid residues, and class III large, heat-labile bacteriocins [17,18]. However, the class I bacteriocins are currently included in the group of ribosomally synthesized and post-translationally modified peptides (RiPPS) [18,19,20]. The class II bacteriocins can be further divided into the subclasses pediocin-like (class IIa), two-peptide (class IIb), leaderless (class IIc) and the non-pediocin single (class IId) bacteriocins [18,21]. In most cases, bacteriocin production appears to be regulated and involves a quorum-sensing mode of regulation [22,23]. The production of nisin is autoregulated by NisK, a sensor kinase protein, and NisR, a response regulator, which regulate transcription via signal transduction by a two-component regulatory system. The induction of NisA or NisZ is likely to be dependent on their interaction with NisK [23]. The NisI and the ATP-binding cassette transporter NisEFG support the immunity of the producer cells to nisin [24].
The presence of LAB in many ecological niches, including fish and their rearing environment, is well characterized, and the antimicrobial activity and the probiotic potential of the isolated strains is emphasized [25,26,27,28,29]. However, despite the beneficial role of most LAB, the species Lactococcus garvieae has been identified as the aetiological agent of lactococcosis, a major, re-emerging and widely distributed ichthyopathology that causes a hyperacute haemorrhagic septicaemia and relevant economic losses in rainbow trout (Oncorhynchus mykiss, Walbaum) farming [25,30]. The reported human cases of pathologies associated with this bacterium are increasing, and its zoonotic potential has been accepted [31,32,33].
Bacteriocinogenic L. garvieae that demonstrate an antagonistic activity against virulent L. garvieae strains have been isolated. Their bacteriocins have been characterized, such as garvicin A [34], garvicin AG1 and garvicin AG2 [35], garvicin KS [36], garvicin L1-5 [37], garvicin ML [38] and garvieacin Q [39]. GarA, produced by L. garvieae 21881, is encoded in the plasmid pGL5, which holds the structural gene of GarA (lgnA), its putative immunity protein (lgnI) and the ABC-transporter and its accessory proteins (lgnC and lgnD). GarA is synthesized as a 63 amino acid precursor with a typical double-glycine leader peptide. Once processed, this peptide results in a 43-aa mature peptide with a theoretical molecular mass of 4645.5 Da. GarA has a narrow activity spectrum and is limited only to other L. garvieae strains, suggesting that its mechanism of action is based on the inhibition of cell division, most likely by inhibiting septum formation in target cells [34]. GarQ is produced by L. garvieae BCC 43588 and is encoded as the GarQ structural gene (garQ), its putative immunity protein (garI) and the ABC-transporter (garT). GarQ shows a wide spectrum of antimicrobial activity against LAB and other potentially foodborne pathogenic strains [39]. GarQ is synthesized as a 70-aa precursor with a double-glycine cleavage site, resulting in mature GarQ of 50-aa and a theoretical molecular mass of 5340 Da. GarQ utilizes the IID and IIC subunits of the mannose phosphotransferase system (Man-PTS) as a receptor. It kills target bacteria by disrupting the membrane integrity, mainly locking the Man-PTS into a conformation that leads to the formation of a constitutively open pore [40,41]. NisZ, a 34 amino acid pentacyclic peptide naturally produced by L. lactis, exhibits antimicrobial activity against several Gram-positive and Gram-negative bacteria [42], and the bacteriocin exerts its antimicrobial activity by both pore formation and the inhibition of cell wall synthesis through specific binding to lipid II, which is an essential precursor of the bacterial cell wall [43].
The cloning and heterologous expression of bacteriocins by LAB, particularly Lactococcus lactis, has proven to be a promising approach for obtaining microbial cell factories with a potent antimicrobial activity [44,45,46,47,48]. Moreover, the simultaneous production of bacteriocins of different classes and/or subclasses and distinct modes of action may not only improve their antimicrobial activity and spectrum in a synergistic fashion but may also reduce the presence of bacteria that are resistant to their antagonistic activity [49,50,51]. In this study, we have proceeded to the design and expression in different L. lactis strains of up to three different bacteriocins with antimicrobial activity against virulent L. garvieae. These bacteriocins, namely, GarA, GarQ and NisA/NisZ, show different modes of action and other well-described beneficial effects, such as the anticarcinogenic effect of NisA/NisZ and its ability to modulate the microbiota and regulate the immune system of its host. Thus, synthetic genes that encode the signal peptide of the lactococcal secreted protein Usp45 (SPusp45), fused to either mature GarA (lgnA) with its putative immunity gene (lgnI) and/or to mature GarQ (garQ) with its immunity gene (garI), were cloned into the protein expression vectors pMG36c which encodes the P32 constitutive promoter and in plasmid pNZ8048c, under control of the inducible PnisA promoter. Recombinant L. lactis strains were then obtained, and their antimicrobial activity against virulent L. garvieae was determined.

2. Materials and Methods

2.1. Bacterial Strains, Plasmids and Growth Conditions

The bacterial strains and plasmids used in this study are listed in Table 1. The L. lactis strains were grown at 30 °C in M17 broth (Oxoid Ltd., Basingstoke, UK) supplemented with 0.5% (w/v) glucose (GM17). Pediococcus damnosus CECT4797 was grown in MRS broth (Oxoid Ltd.) at 30 °C. Escherichia coli JM109 (Promega, Madison, WI, USA) was grown in Luria–Bertani (LB) broth (Oxoid Ltd.) at 30 °C with shaking. Chloramphenicol (Sigma-Aldrich, St. Louis, MO, USA) was added at 20 µg/mL to select growth of E. coli and at 5 µg/mL for the selection of the recombinant lactococcal strains. The cell dry weights of the late exponential phase cultures were determined gravimetrically. Agar plates were made by the addition of 1.5% (w/v) agar (Oxoid) to the liquid media.

2.2. Basic Genetic Techniques and Enzymes

Synthetic gene fragments were designed from the described amino acid sequence of the bacteriocin GarA (lgnA) and GarQ (garQ), as well as those from their putative immunity proteins GarAI (lgnI) and GarQI (garI), respectively. In addition, the leader peptide of the native bacteriocins was replaced by the signal peptide of the secreted protein Usp45 (SPusp45), a Sec-dependent protein produced by L. lactis MG1363 [45,57]. Similarly, additional sequences containing the SacI cleavage site and the P32 ribosome binding site (RBS) as well as the SacI/HindIII or the BspHI/HindIII enzyme restriction cleavage sites were added at the 5′ and 3′ ends, respectively, of the designed synthetic gene fragments. Their codon usage was adapted for its expression by L. lactis. GeneArt® supplied the synthetic genes into the carrier plasmid pMA-T (Life Technologies S.A., Madrid, Spain). The protein expression vectors pMG36c and pNZ8048c were purified from E. coli JM109 by using the NucleoSpin Plasmid Kit (Macherey-Nagel, Düren, Germany). DNA restriction enzymes were supplied by New England Biolabs (Beverly, MA, USA). Ligations were performed with the T4 DNA ligase (Invitrogen, Walthman, MA, USA). Electrocompetent L. lactis subsp. cremoris NZ9000, L. lactis subsp. cremoris WA2-67, L. lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24 cells were obtained after successive growth in the SGGM17 medium, which consisted of of M17 (Oxoid Ltd.) supplemented with 0.5 M sucrose, glucose (0.5%; w/v) and glycine (2%; w/v). The cultures were centrifuged and resuspended in a cold wash buffer containing glycerol (20%; v/v) and 0.5 M sucrose. Aliquots of 50 µL were stored at −80 °C until further use.

2.3. Recombinant Plasmids Derived from pMG36c and Transformation into L. lactis Hosts

The primers and PCR products used for the construction of the pMG36c-derived vectors are listed in Table S1. PCR product amplifications were performed in 50 μL reaction mixtures that contained 20 ng of the synthetic gene fragments included in the carrier pMA-T vectors, 70 pmol of each primer, 1 U of Velocity DNA polymerase (Bioline Reagents, Ltd., London, UK), 10 µL of Hi-Fi buffer and 1.5 µL of 30 mM dNTP’s mix in a MJ Mini Gradient Thermal Cycler (BioRad Laboratories). PCR cycling conditions were as follows: denaturation at 98 °C (2 min), 35 cycles of denaturation–annealing–extension (98 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, respectively) and a final extension step at 72 °C (5 min). The PCR-generated fragments were purified using a NucleoSpin® Gel and PCR clean-up kit (Macherey-Nagel) for cloning and nucleotide sequencing. When required, PCR amplifications were sequenced 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 (CAI Técnicas Biológicas, UCM, Madrid, Spain). Digestion of the amplified PCR products with SacI/HindIII permitted the ligation of the resulting restriction fragments into pMG36c, which was digested with the same enzymes. The resulting pMG36c-derived vectors were transformed into competent lactococcal hosts and electrotransformed with a Gene PulserTM and Pulse Controller apparatus (Bio-Rad Laboratories, Hercules, CA, USA), according to a previously described procedure [58]. Transformed cells containing the pMG36c-derived vectors pJFAI, pJFQI, pJFAIQI and pJFQIAI (Table 1), were selected for their growth with chloramphenicol and evaluated for their bacteriocinogenity. The total bacterial DNA from the transformed lactococcal strains was purified using the InstaGene Matrix (BioRad Laboratories). It was then submitted to PCR using the primers MGPJ-F and MGPJ-R. The sequencing of the generated PCR products was performed at the Unidad de Genómica (CAI Técnicas Biológicas, UCM).

2.4. Recombinant Plasmids Derived from pNZ8048c and Transformation into L. lactis Hosts

The primers and PCR products used for the construction of the pNZ8048c-derived vectors are listed in Table S1. The initial PCR amplification of the designed gene fragments into carrier pMA-T vectors was performed with primers GARF-BSPHI and GARAIM-R, which were designed to provide the restriction cleavage site for BspHI/HindIII. The digestion of the amplified PCR products with BspHI/HindIII permitted the ligation of the resulting restriction fragments into pNZ8048c, which was digested with NcoI and HindIII. However, it should be highlighted that construction of the pNZ8048c-derived vectors carrying GarQ or GarQ and GarA and their respective immunity proteins was achieved using a novel, PCR-based, restriction-enzyme-free cloning, or ABC cloning, method [59]. Briefly, the procedure implies the PCR amplification of three overlapping fragments, two from the pNZ8048c vector and one from the previously designed synthetic gene fragments, to generate a single, circular, pNZ8048c-derived vector by using a pair of overlapping primers. For the amplification of the appropriate gene fragments, 50 µL PCR reactions containing 100 ng of plasmid pNZ8048c or the synthetic gene fragments included in the carrier pMA-T vectors, 0.5 µmol of each primer and 25 µL of Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Scientific, Waltham, MA, USA) were used. PCR cycling conditions were as follows: one initial denaturation step at 98 °C (30 s), 30 cycles of denaturation–annealing–extension (98 °C for 10 s, 49.1–65.8 °C for 20 s and 72 °C for 25 s, respectively) and a final extension step at 72 °C for 5–10 min). Overlapping PCRs were carried out using the three corresponding fragments as templates. Specifically, 1.5 × 1010 copies of fragments derived from amplification of pNZ8048c and 3 × 1010 copies of fragments obtained from amplification of the designed synthetic genes were used. Agarose gel electrophoresis, visualization and sequencing of the generated PCR products were performed essentially as described for the construction of the pMG36c-derived vectors. The resulting pNZ8048c-derived vectors were transformed into competent lactococcal hosts, and the transformed cells containing the pNZ8048c-derived vectors pNJFAI, pNJQI and pNJFQIAI (Table 1) were selected for their growth with chloramphenicol and evaluated for their bacteriocinogenicity. Bacterial DNA from the lactococcal transformed cells was submitted to PCR amplification with the primers NZPJ-F and NZPJ-R. The sequencing of the generated PCR products was performed at the Unidad de Genómica (CAI Técnicas Biológicas, UCM).

2.5. Antimicrobial Activity of the Recombinant L. lactis Strains

The direct antimicrobial activity of colonies from the recombinant lactococcal strains was examined by a stab-on-agar test (SOAT) as previously described [60]. When appropriate, cultures were induced with nisin A (Sigma-Aldrich) at a final concentration of 10 ng/mL for the production of the cloned bacteriocins. Cell-free culture supernatants (CFS) were obtained by the centrifugation of cultures at 12,000× g at 4 °C for 10 min, adjusted to pH 6.2 with 1 M NaOH, filtered through 0.22 μm pore-size syringe filters (Sartorius, Göttingen, Germany) and stored at −20 °C until further use. The antimicrobial activity of the supernatants was determined by an agar diffusion test (ADT). It was further quantified by a microtiter plate assay (MPA) as previously described [60]. For the MPA, the growth inhibition of sensitive cultures was measured spectrophotometrically at 620 nm with a FLUOstar OPTIMA (BMGLabtech, Ortenberg, Germany) plate reader. One bacteriocin unit (BU) was defined as the reciprocal of the highest dilution of the bacteriocin that caused a growth inhibition of 50% (50% of the turbidity of the control culture without bacteriocin).

2.6. Purification of Bacteriocins

Bacteriocins were purified as previously described using a multi-chromatographic procedure [60]. Briefly, 1 L supernatants from early stationary cultures of the recombinant lactococci were precipitated with (NH4)2SO4 (50%; w/v), desalted by gel filtration (PD-10 columns) and subjected to a cation-exchange (SP Sepharose Fast Flow), followed by a hydrophobic interaction (Octyl-Sepharose CL-4B) and reverse-phase chromatography in an ÄKTA purifier Reverse Phase Fast Protein Liquid Chromatography system (RP-FPLC), using the PepRPC HR 5/5 column. Fractions exhibiting the highest bacteriocin activity were pooled and re-chromatographed on the same column until chromatographically pure bacteriocin peptides were obtained. All chromatographic columns and equipment were obtained from GE Healthcare Life Sciences (Barcelona, Spain).

2.7. Mass Spectrometry (MS) and Multiple Reaction Monitoring (MRM) Analysis of Purified Peptide Fractions from Supernatants of the Recombinant L. lactis Strains

Purified RP-FPLC fractions from the supernatants of the recombinant lactococcal strains were subjected to matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) and multiple reaction monitoring liquid chromatography–electrospray ionization tandem mass spectrometry (MRM-LC-ESI-MS/MS) analyses at the Unidad de Proteómica (CAI Técnicas Biológicas, UCM). Briefly, 1 μL of eluted fractions were spotted onto a MALDI target plate and allowed to air-dry at room temperature. Then, 0.8 μL of a sinapic acid matrix (Sigma-Aldrich) in 30% acetonitrile and 0.3% trifluoroacetic acid was added and allowed to air-dry at room temperature. MALDI-TOF MS analyses were performed using a 4800 Plus Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada).
For the identification of bacteriocins, the MRM method evaluates a complex mixture of tryptic peptides that can be selectively detected by liquid chromatography coupled to electrospray MS. Briefly, the purified RP-FPLC fractions of interest were dried in Speed-vac and resuspended in 20 μL of 8 M urea. The samples were reduced by adding 10 mM of dithiothreitol for 45 min at 37 °C and alkylated with 55 mM of iodacetamide for 30 min in the dark. The urea was then diluted with 25 mM of ammonium bicarbonate to obtain a molarity of less than 2. When the pH was 8.5, digestion was performed by adding recombinant sequencing grade Trypsin (Roche Molecular Biochemicals, Branchburg, NJ, USA) 1:20 (w/w) and incubating at 37 °C. After 60 min, an aliquot was taken for the partial digestion of the sample. The rest was incubated overnight. The produced peptides were dried in Speed-vac and resuspended in 2% acetonitrile and 0, 1% formic acid. Skyline (64-bit), version 20.1, was used to build and optimize the MRM for the detection of the peptides of interest [61].
All analyses were performed on a LC-MS/MS Eksigent Nanoflow LC system coupled to a hybrid triple quadrupole/ion trap mass spectrometer, 5500 QTRAP (AB Sciex, Foster City, CA, USA), equipped with a nano electrospray interface operating in the positive ion mode. The MS/MS data were analyzed using Protein Pilot 4.5 software (AB Sciex) or MASCOT 2.3 (MatrixScience, London, UK) to identify the peptides against an in-house DataBase with the fasta sequences of the targeted proteins. The searches were performed assuming a digestion with trypsin with a maximum of 2 missed cleavages, a fragment ion mass tolerance of 0.6 Da and a parent ion tolerance of 0.15 Da. Peptide identifications based on the MS/MS data were accepted if they could be established at a CI of greater than 95% (p < 0.05). Data were then processed against the MRM-library on Skyline to ensure consistency between the transitions detected and the sequences of the peptides searched.

3. Results

3.1. Genetic Design and Cloning of Synthetic Genes That Drive the Heterologous Production of GarA and/or GarQ by Recombinant L. lactis Cells

In this work, synthetic genes containing the protein SPusp45, fused to mature GarA (lgnA) and its putative immunity protein GarAI (lgnI) (AI) encoded by L. garvieae 21881 [34], synthetic genes containing the protein SPusp45 fused to mature GarQ (garQ) and its putative immunity protein GarQI (lgnI) (QI) encoded by L. garvieae BCC43578 [39], and synthetic genes containing the genetic fusions SPusp45::lgnA+lgnI+garQ+garI (AIQI) and SPusp45::garQ+garI+lgnA+lgnI (QIAI), were designed for cloning into the protein expression vectors pMG36c which carries the P32constitutive promoter, and in pNZ8048c with the inducible PnisA promoter. PCR-based amplifications of the synthesized gene fragments allowed for the generation of the PCR products shown in Table S1. Cloning the PCR products A, B, C and D in pMG36c resulted in the pMG36c-derived vectors pJFAI, pJFQI, pJFAIQI and pJFQIAI, and cloning the PCR product E in pNZ8048c resulted in the pNZ8048c-derived vector pNJFAI (Table 1). Similarly, the use of a novel, PCR-based, restriction-enzyme-free cloning method [59] for cloning fragments in plasmid pNZ8048 allowed for the construction of the pNZ8048c-derived vectors pNJFQI and pNJFQIAI (Table 1). In this study, no efforts were made to omit the presence of hypothetically redundant genes that encode putative immunity proteins in the designed synthetic gene fragments.

3.2. Antimicrobial Activity of the Recombinant L. lactis Strains as Determined by Their Direct Antagonistic Effect (SOAT) and the Antimicrobial Activity (ADT) of Their Cell-Free Supernatants

The transformation of recombinant plasmids into L. lactis subsp. cremoris NZ9000 showed that while the control NZ9000 (pMG36c) and NZ9000 (pNZ8048c) cells showed no antimicrobial activity against L. garvieae CF00021 or P. damnosus CECT4797, all the recombinant NZ9000-derived strains exhibited a measurable antagonistic effect against L. garvieae CF00021 (Table 2). Interestingly, results from both the SOAT and ADT tests showed that the recombinant strains NZ9000 (pJFAI) and NZ9000 (pNJFAI), which only encode the production of GarA, showed no antimicrobial activity against P. damnosus CECT4797.
However, the recombinant strains derived from the native NisZ producer (L. lactis subsp. cremoris WA2-67) and the native NisA producers (L. lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24) showed a higher antimicrobial activity against P. damnosus CECT4797 than L. garvieae CF00021. The strains that only encoded the production of GarA and/or GarQ but not NisZ or NisA (NZ9000 derivatives) showed a weaker antimicrobial activity against P. damnosus CECT4797. A closer evaluation of the antimicrobial activity of the NisZ-producing strain L. lactis subsp. cremoris WA2-67 suggested that the strains WA2-67 (pJFQI) and WA2-67 (pJFQIAI) displayed slightly larger halos of inhibition against both indicator strains than the rest of the recombinant strains evaluated (Table 2). Furthermore, the evaluation of the diluted supernatants of L. lactis subsp. cremoris WA2-67, which were transformed with pMG36c or the pMG36c-derived vectors, showed that the antimicrobial activity of the recombinant strains WA2-67 (pJFAI), WA2-67 (pJFQI), WA2-67 (pJFAIQI) and WA2-67 (pJFQIAI) was 2-, 8-, 2- and 16-fold higher, respectively, than the antimicrobial activity of L. lactis subsp. cremoris WA2-67 (pMG36c) (Figure 1).
The evaluation of the antimicrobial activity of the NisA-producing strain L. lactis subsp. lactis DPC5598 showed that recombinants encoding the production of either GarAI, GarQI, GarAI+GarQI or GarQI+GarAI displayed similar halos of inhibition to the control strains, DPC5598 (pMG36c) and DPC5598 (pNZ8048c), against both P. damnosus CECT4797 and L. garvieae CF00021. However, supernatants of the recombinant strains, derived from the NisA producer L. lactis subsp. lactis BB24, showed slightly larger inhibition halos against L. garvieae CF00021 than the control BB24 (pMG36c) and BB24 (pNZ8048) cells (Table 2).

3.3. Antimicrobial Activity of Recombinant L. lactis Strains against Different L. garvieae Strains

The antimicrobial activity of supernatants from all lactococcal strains was quantified against different virulent L. garvieae strains by using a more sensitive microtiter plate assay (MPA). Again, L. lactis subsp. cremoris NZ9000 (pMG36c) showed no antimicrobial activity against any of the L. garvieae strains evaluated, while the recombinant NZ9000-derived strains transformed with the constitutive pMG36c-derived vectors showed a weak antimicrobial activity against the virulent L. garvieae strains (Table 3). However, the NisZ-producing L. lactis subsp. cremoris WA2-67 (pMG36c) showed a much higher antimicrobial activity against L. garvieae. Remarkably, the antimicrobial activity determined for the recombinant WA2-67-derived strains transformed with the pMG36c-derived vectors to generate the WA2-67 (pJFAI), WA2-67 (pJFQI), WA2-67 (pJFAIQI) and WA2-67 (pJFQIAI) bacteriocin producers was 1.0- to 1.6-fold, 5.1- to 10.7-fold, 0.9- to 1.6-fold, and17.3- to 68.2-fold higher, respectively, than that of the control strain WA2-67 (pMG36c) (Table 3). The antimicrobial activity of the NisA producer L. lactis subsp. lactis DPC5598 recombinants was 0.5- to 1.2-fold higher than that of the control DPC5598 (pMG36c) strain against L. garvieae, while the antagonistic activity of the NisA producer L. lactis subsp. lactis BB24 recombinants was 1.3- to 3.7-fold higher than that of the control BB24 (pMG36c) strain against the same L. garvieae indicator strains (Table 3).
When the antimicrobial activity of the lactococcal strains that were transformed with the inducible pNZ8048c-derived vectors was determined, L. lactis subsp. cremoris NZ9000 (pNZ8048c) showed no antimicrobial activity against any of the L. garvieae strains, while the recombinant NZ9000 (pNJFAI), NZ9000 (pNJFQI), and NZ9000 (pNJQIAI) strains showed a 12.8- to 18.9-fold, 7.4- to 18.1-fold and 6.9- to 14.4-fold higher antimicrobial activity, respectively, than the cells transformed with the pMG36c-derived vectors (Table 4). However, the antimicrobial activity of the L. lactis subsp. cremoris WA2-67 (pNZ8048c) cells and their recombinant WA2-67 (pNJFAI), WA2-67 (pNJFQI) and WA2-67 (pNJFQIAI) strains showed a 0.7- to 0.9-fold, 0.5- to 0.8-fold, 0.1- to 0.2-fold and 0.01- to 0.08-fold lower antimicrobial activity, respectively, against the L. garvieae strains than the cells transformed with the pMG36c-derived vectors (Table 4). The antimicrobial activity of the NisA-producing L. lactis subsp. lactis DPC5598 transformed with the pNZ8048c-derived vectors was only slightly (0.9- to 2.0-fold) higher than the antimicrobial activity of the cells transformed with the pMG36c-derived vectors. Similarly, the antagonistic activity of the NisA producer L. lactis subsp. lactis BB24 recombinants transformed with the pNZ8048c-derived vectors was only slightly (0.6- to 1.9-fold) higher than cells transformed with the pMG36c-derived vectors (Table 4).

3.4. Purification of Bacteriocins Produced by L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI)

The results regarding the purification to homogeneity of the bacteriocins in supernatants of the selected L. lactis subsp. cremoris recombinants are summarized in Table 5. The evaluation of the most active antimicrobial fractions after the first reversed-phase chromatography step (RP-FPLC) permitted the identification of two active fractions during the purification of the bacteriocins produced by L. lactis subsp. cremoris WA2-67 (pJFQI) and three active fractions during the purification of the bacteriocins produced by L. lactis subsp. cremoris WA2-67 (pJFQIAI). Although the antimicrobial activity of the eluted fractions was low, a significant increase in their specific antimicrobial activity was observed.

3.5. Mass Spectrometry (MS) and Multiple Reaction Monitoring (MRM) Analysis of the Purified Bacteriocin Fractions

MALDI-TOF MS analysis of fraction 8 and fraction 7, eluted during the RP-FPLC step of the purification of supernatants from L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI), showed major peaks of 3331.4 Da and 3331.3 Da (Figure S1), respectively, matching the molecular mass described for NisZ. In addition, second peaks of 3349.3 Da and 3348.8 Da, respectively, likely correspond to the oxidation of the lanthionine ring of NisZ (Figure S1).
However, MALDI-TOF MS analysis of the eluted fraction 14 from the L. lactis subsp. cremoris WA2-67 (pJFQI), which encoded GarQ and NisZ, in addition to an analysis of fractions 9 and 12 from the L. lactis subsp. cremoris WA2-67 (pJFQIAI), which encoded GarA, GarQ and NisZ, could not identify the presence of the bacteriocins GarA and GarQ with predicted molecular masses of 4645.2 Da and 5340 Da, respectively, in the eluted fractions. Since this was a totally unexpected result, the fractions were subjected to MRM-LC-ESI-MS/MS analysis to determine the presence of the expected bacteriocins in the samples. In the MRM method, a series of target tryptic peptides and their associated transitions (fragments m/z) were predicted from the molecular masses and amino acid sequences of the bacteriocins GarA and GarQ. Each targeted peptide has a set of accompanying transitions which are then selectively detected in the second stage of the MS. A summary of the results obtained is shown in Table 6. MRM transitions were established and validated by tandem mass spectrometry (MS/MS). For each bacteriocin, two encrypted peptides were confidently detected in duplicate runs.

4. Discussion

Virulent L. garvieae are the etiological agents of a hyperacute hemorrhagic septicemia in fish, known as lactococcosis. They are also responsible for human pathologies due to their zoonotic character and potential presence in foods [31,33]. Bacteriophages and bacteriocins have potential as complementary strategies for combating L. garvieae in foods and fish [26,27], while bacteriocinogenic LAB could be evaluated for their potential use as probiotics, paraprobiotics and postbiotics in food, feed and other biotechnological applications [7,53,62]. The optimization of bacteriocin gene synthesis, expression and production helps the development of LAB as cell factories for the production and delivery of multiple bacteriocins [45,46,63]. The use of synthetic genes that match the codon usage of the producer organisms has a significant impact on gene expression levels and protein folding [47,64].
In this work, the transformation of L. lactis subsp. cremoris NZ9000 with pMG36c- or pNZ8048c-derived vectors demonstrated that the NZ9000 (pJFAI) and NZ9000 (pNJFAI) recombinant cells, which encode GarAI, showed antimicrobial activity against L. garvieae CF00021 but not against P. damnosus CECT4797 (Table 2), confirming their production of GarA and previous observations that this bacteriocin was only active against L. garvieae [34]. However, differences in the antimicrobial activity of recombinants derived from the NisZ producer L. lactis subsp. cremoris WA2-67, which was transformed with the pMG36c-derived vectors but not pNZ8048c-derived vectors, were observed against both indicator strains. The obtained results showed that the WA2-67 (pJFQI) cells exhibited larger halos of inhibition than the WA2-67 (pJFAI) cells, and that the WA2-67 (pJFQIAI) cells displayed the largest observed halos (Figure 1). These results suggest that the constitutive expression of GarQI is higher than GarAI or that the specific antimicrobial activity of GarQ is higher than that of GarA. Perhaps the transcription, processing and secretion from genes encoding GarQI+GarAI are more effective than from genes encoding GarAI+GarQI. On the other hand, no remarkable differences were found in the antimicrobial activity of the recombinants derived from the NisA producers transformed with the pMG36c-derived or the pNZ8048c-derived vectors, L. lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24 (Table 2).
Due to the increasing interest that L. garvieae is attracting as not only a relevant bacterial pathogen but also as a zoonotic agent [29,65,66], the antimicrobial activity of the L. lactis recombinants was further evaluated and quantified against different virulent L. garvieae strains by using a more sensitive microplate assay (MPA). The obtained results showed that the L. lactis subsp. cremoris NZ9000 cells transformed with the pNZ8048c-derived vectors showed a 6.9- to 18.9-fold higher antimicrobial activity than the recombinant cells bearing the pMG36c-derived vectors (Table 3 and Table 4). The enhanced antimicrobial activity in cells with the nisin-inducible constructs may be due to copy number differences between pNZ8048c and pMG36c, but is more likely caused by the promoters used to drive gene expression [67,68]. Plasmid pNZ8048c contains the high-copy number heterogramic replicon of the lactococcal plasmid pSH71 with a unique NcoI cleavage site downstream of the nisA ribosome binding site (RBS), which is used for translational fusions inducible by NisA [23,69]. To optimize protein production, inducible systems are usually considered superior to constitutive expression systems since the former allow for the achievement of a sufficient biomass before the initiation of target protein expression [70]. The increased antimicrobial activity observed with the NisA-induced cells may also be ascribed to the short induction time for the production of GarA and/or GarQ (3 h), which most likely prevented the secreted bacteriocins from attaching to cell walls to form aggregates and/or to undergo protease degradations.
Moreover and quite remarkably, the antimicrobial activity of L. lactis subsp. cremoris WA2-67 (pMG36c) and the pMG36c-derived WA2-67 (pJFAI), WA2-67 (pJFQI), WA2-67 (pJFAIQI), and WA2-67 (pJFQIAI) strains showed a 1.0- to 1.6-fold, 5.1- to 10.7-fold, 0.9- to 1.6-fold and 17.3- to 68.2-fold higher antimicrobial activity, respectively, than the control WA2-67 (pMG36c) strain (Table 3). These results also indicate that the expression of QI increases the antimicrobial activity of the producer cells; however, the expression of AI has a much lower effect. Additionally, no increase in antimicrobial activity is observed when QI is expressed as the second of the two modules (AIQI). However, when QI is expressed as the first module (QIAI), a synergistic effect of both modules seems to occur regarding the very high antimicrobial activity of the producer cells. Thus, the AI in the AIQI module appears to prevent the QI from becoming active. However, when QI is expressed first in the QIAI module, the AI appears to synergistically increase the QI activity (Table 3). The pMG36c vector is a shuttle vector. It is based on the low-copy replication origin of pWV01 and is able to replicate in Escherichia coli, Bacillus subtilis and LAB, whereas the strong P32 promoter drives the constitutive transcription of inserted genes into the multicloning site (MCS) of pUC18 [56]. From the results obtained, it may occur that, as previously suggested, the specific antimicrobial activity of GarA against L. garvieae is lower and/or its production and stability is less than that of GarQ. Additionally, besides the choice of vector and promoters, other factors such as the activation of quality control networks involving folding factors and housekeeping proteases, the oxidation of methionine to methionine-sulfoxide, bacteriocin self-aggregation and mRNA stability may affect bacteriocin production and activity from the recombinant hosts [46,71]. The coexpression of putative immunity genes may also increase the production of bacteriocins in heterologous hosts. These immunity proteins can act by either affecting bacteriocin pore formation or by perturbing the interaction between the bacteriocin and a membrane-located bacteriocin receptor, thereby preventing producer cells from being killed [16]. The expression in AIQI of LgnI before the expression of GarI could also affect producer protection against GarQ, thereby affecting growth and bacteriocin production by the producer cells. Importantly, L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI) showed the highest antimicrobial activity against all virulent strains of L. garvieae evaluated (Table 3).
However, the antimicrobial activity of L. lactis subsp. cremoris WA2-67, which was transformed with the pNZ8048c-derived vectors, produced WA2-67-derivatives that showed a 0.01- to 0.9-fold lower antimicrobial activity, respectively, than the cells transformed with the pMG36c-derived vectors (Table 4). This was an unexpected result since, as previously described, inducible systems are often considered superior to constitutive expression systems for the optimization of protein production. Perhaps NisK and NisR, the two component signal transduction systems for the regulation of NisZ synthesis in L. lactis subsp. cremoris WA2-67, do not fully activate transcription of the PnisA present in pNZ8048c. It could be also possible that levels of phosphorylated NisR may be not enough to drive the activation of two independent Pnis promoters which, in addition, derive from two different Lactoccocus lactis subspecies: cremoris and lactis, respectively. Alternatively, NisZ may not be as efficient as NisA for an interaction with the NisK produced by L. lactis subsp. cremoris WA2-67, thus constraining the induction of transcription of PnisA in pNZ8048c by blocking NisZ with NisI and NisEFG.
The antimicrobial activity of the NisA-producers L. lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24 was slightly higher for the cells transformed with the pNZ8048c- than the pMG36c-derived vectors, suggesting that NisA is a better inducer than NisZ for the activation of the transcription of PnisA in pNZ8048c (Table 3 and Table 4). However, the antimicrobial activity of the DPC5598-derived recombinants under constitutive or inducible conditions was lower during multi-bacteriocin production. This was probably due to the high energy and metabolic cost linked to plasmid maintenance and replication, to the secretion stress associated with bacteriocin overproduction and/or to the synthesis of proteinases for the elimination of misfolded proteins [72,73,74]. Differences in the antimicrobial activity of these strains and those from the L. lactis subsp. cremoris WA2-67 transformed with the pMG36c-derived vectors may be also ascribed to yet-unknown genetic and/or metabolic differences between the strains. L. lactis subsp. lactis DPC5598 was selected as a potential multi-bacteriocin-producing host because it is a plasmid-free derivative of an industrial strain that is extensively used in fermented dairy products due to its phage insensitivity and fast acid-producing ability [54]. L. lactis subsp. lactis BB24 is a fermented, meat-derived isolate widely used as an efficient host for the heterologous production of bacteriocins [45,55]. Both multi-bacteriocin producers should be considered for their potential evaluation as probiotics, paraprobiotics and/or postbiotics reducing the increasing presence of virulent and zoonotic L. garvieae in selected milk and meat substrates, respectively [31,75].
The MALDI-TOF MS analysis of purified eluted fractions from supernatants of the most active antimicrobial strains, L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI) (Table 5), allowed for the detection of NisZ in supernatants of the producer strains (Figure S1), suggesting that this bacteriocin is appropriately processed and transported out of the producer cells. However, the presence of GarA and GarQ could not be detected, suggesting interactions of the bacteriocins with unknown biological compounds, or a low amount and recovery of the bacteriocins during their purification to homogeneity [47,64]. However, bacteriocins in the purified fractions were suitable for MRM evaluation and data analysis, which is an emerging targeted proteomics workflow and a highly selective and sensitive method for detecting peptides in the low ng/mL to sub-ng/mL range concentrations [64,76,77]. When the purified fractions were subjected to MRM-LC-ESI-MS/MS analysis, two encrypted peptides were confidently (99%) detected. The detected peptides were confirmed by MS/MS, and at least four transitions were identified for each (Table 6). The peptide fragments covered 44% of the sequence of GarQ, while the coverage was 21% for GarA. These relatively low-coverage percentages are related to the reduced presence of lysine and arginine (trypsin cleavage sites) in GarA and GarQ, which significatively reduces the number of potentially identifiable target peptides.
Previous studies by our research group identified probiotic features of the native or wild-type NisZ producer L. lactis subsp. cremoris WA2-67 such as a potent antimicrobial activity against ichthyopathogens, survival in fresh water and the gastrointestinal tract of trout, a resistance to bile and low pH, and an improved colonization ability with respect to the intestinal trout mucosa [26,53,78]. Further in silico analyses of the whole-genome sequence (WGS) of this strain also identified other potential probiotic traits such as the production of vitamins and amino acids, adhesion/aggregation and stress resistance factors and the absence of transferable antibiotic resistance determinants and genes encoding detrimental enzymatic activities or potential virulence factors [79]. Other studies performed by our group demonstrated the effectiveness of L. lactis subsp. cremoris WA2-67 to protect rainbow trout in vivo against infection of the virulent L. garvieae and the relevance of NisZ production as an anti-infective mechanism [53].
The work described in this study constitutes the first report on the design of multi-bacteriocinogenic L. lactis subsp. cremoris WA2-67 strains with a high antimicrobial activity against virulent L. garvieae and a promising role as probiotics, paraprobiotics and/or postbiotics in food, feed and other biotechnological applications. The evaluation of bioengineered strains as probiotics is subjected to approval by regulatory authorities and is performed under strict biological conditions. However, the number of reports on the evaluation of bioengineered bacterial strains as probiotics (live cells), paraprobiotics (dead, non-viable cells) and postbiotics (physical-, chemical- or enzymatic-lysis of probiotic cells) is increasing [7,80,81]. Accordingly, experiments are being planned to evaluate the in vitro effect of L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI) on rainbow trout intestinal epithelial cells (RTgutGC) for a transcriptional analysis of several immune, intestinal, barrier-integrity and homeostasis genes and the induction of antimicrobial peptides (AMPs), as well as for their effect on the in vivo modulation of the intestinal microbiota and immune response of rainbow trout (Oncorhynchus mykiss, Walbaum) and turbot (Scophthalmus maximus).

5. Conclusions

The design of synthetic genes and their cloning into protein expression vectors bearing constitutive or inducible promoters has allowed for the production and functional expression of GarA and/or GarQ by L. lactis subsp. lactis and L. lactis subsp. cremoris strains. Most importantly, L. lactis subsp. cremoris WA2-67, transformed with the pMG36c-derived vectors, allowed for the obtention of L. lactis subsp. cremoris WA2-67 (pJFQI), a producer of GarQ and NisZ, and L. lactis subsp. cremoris WA2-67 (pJFQIAI), a producer of GarA, GarQ and NisZ, with a much higher antimicrobial activity (5.1- to 10.7-fold and 17.3- to 68.2-fold, respectively) against virulent L. garvieae than the rest of the L. lactis strains evaluated. The concerted use of a sensitive microtiter plate assay (MPA) for the quantification of the antimicrobial activity of supernatants, the use of a multi-chromatographic procedure for the purification of bacteriocins to homogeneity, and the use of a MALDI-TOF MS multiple reaction monitoring (MRM-LC-ESI-MS/MS) analysis of the purified bacteriocins are unavoidable and possibly irreplaceable tools for the identification and characterization of the bacteriocins produced by L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods12051063/s1, Table S1: Primers and PCR products used in this study; Figure S1: MALDI-TOF MS analysis of purified NisZ from Lactococcus lactis subsp. cremoris WA2-67 (pJFQI) (A) and Lactococcus lactis subsp. cremoris WA2-67 (pJFQIAI (B). Numbers indicate the molecular mass in Daltons of major peptide fragments.

Author Contributions

Conceptualization, P.E.H. and L.M.C.; methodology, J.F., C.A., S.A., E.M.-A. and J.B.; validation, J.F., C.A., S.A., D.C. and B.G.-S.; formal analysis, J.F., L.D.-F., E.M.-A. and J.B.; investigation, J.F., C.A. and S.A.; writing—original draft preparation, J.F.; project administration, P.E.H., L.M.C. and J.F.; funding acquisition, P.E.H. and L.M.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by project RTI2018-094907-B-I00 from the Ministerio de Ciencia, Innovación y Universidades (MCIU, Madrid, Spain), project FEI16/54 from the Universidad Complutense de Madrid (UCM) and project PR75/18–21627 from Santander/UCM. Javier Feito was supported by a FEI16/54 contract from UCM and held a predoctoral contract from the UCM. Carlos Araújo and Sara Arbulu held a FEI16/54 contract from the UCM.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article or supplementary material.

Acknowledgments

We thank the Proteomics Unit of the University Complutense of Madrid (UCM), integrated the ProteoRed, PRB3-ISCIII, and support from the Grant PT17/0019 of the PE I + D + I 2013–2016, funded by ISCIII and ERDF, for technical assistance in the proteomic analysis of the samples.

Conflicts of Interest

The authors declare no conflict of interest.

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Foods 12 01063 g001 550
Figure 1. Antimicrobial activity of diluted supernatants of recombinant L. lactis strains as determined by an agar diffusion test (ADT) with L. garvieae CF00021 as the indicator microorganism. Supernatants from: L. lactis subsp. cremoris WA2-67 (pMG36c) (A), L. lactis subsp. cremoris WA2-67 (pJFAI) (B), L. lactis subsp. cremoris WA2-67 (pJFQI) (C), L. lactis subsp. cremoris WA2-67 (pJFAIQI) (D) and L. lactis subsp. cremoris WA2-67 (pJFQIAI) (E). Supernatants from recombinant strains in the upper lines were diluted 1, 2, 4, 8, 16 and 32 times, and in the bottom lines were diluted 64, 128, 256, 512, 1024 and 2048 times.
Figure 1. Antimicrobial activity of diluted supernatants of recombinant L. lactis strains as determined by an agar diffusion test (ADT) with L. garvieae CF00021 as the indicator microorganism. Supernatants from: L. lactis subsp. cremoris WA2-67 (pMG36c) (A), L. lactis subsp. cremoris WA2-67 (pJFAI) (B), L. lactis subsp. cremoris WA2-67 (pJFQI) (C), L. lactis subsp. cremoris WA2-67 (pJFAIQI) (D) and L. lactis subsp. cremoris WA2-67 (pJFQIAI) (E). Supernatants from recombinant strains in the upper lines were diluted 1, 2, 4, 8, 16 and 32 times, and in the bottom lines were diluted 64, 128, 256, 512, 1024 and 2048 times.
Foods 12 01063 g001
Table
Table 1. Bacterial strains and plasmids used in this study.
Table 1. Bacterial strains and plasmids used in this study.
Strain or PlasmidDescription aSource and/or Reference b
Strains
  Lactococcus lactis subsp. cremoris NZ9000Host strain; non-bacteriocin producer; pepN::nisRK.NIZO, [52]
  Lactococcus lactis subsp. cremoris WA2-67Host strain; fish origin; nisin Z producer.SD-
NUTRYCIAL, [53]
  Lactococcus lactis subsp. lactis DPC5598Host strain; milk origin; nisin A producer. Plasmid-free derivative of DPC4268.DPC, [54]
  Lactococcus lactis subsp. lactis BB24Host strain; meat origin; nisin A producer.SD-
NUTRYCIAL, [55]
  Lactococcus garvieae CF00021SOAT, ADT, MPA indicatorCEFAS-BCC
  Lactococcus garvieae CF01144MPA indicatorCEFAS-BCC
  Lactococcus garvieae CLG4MPA indicatorLFP-UNIZAR
  Lactococcus garvieae CLG5MPA indicatorLFP-UNIZAR
  Lactococcus garvieae CLFP28/06MPA indicatorLFP-UNIZAR
  Lactococcus garvieae JIP29/99MPA indicatorJouy-en-Josas SC
  Pediococcus damnosus CECT4797SOAT, ADT indicatorCECT
Plasmids
  pMG36cCmr; pMG36e derivative; constitutive expression vector carrying the P32 promoter.RUG-MG, [56]
  pNZ8048cCmr; inducible expression vector carrying the nisA promoter.NIZO, [52]
  pJFAICmr, pMG36c derivative carrying the PCR product A (P32 ribosome binding site and the SPusp45 fused to mature lgnA and lgnI).This work
  pJFQICmr, pMG36c derivative carrying the PCR product B (P32 ribosome binding site and the SPusp45 fused to mature garQ and garI).This work
  pJFAIQICmr, pMG36c derivative carrying the PCR product C (P32 ribosome binding site and the SPusp45 fused to mature lgnA and lgnI and mature garQ and garI).This work
  pJFQIAICmr, pMG36c derivative carrying the PCR product D (P32 ribosome binding site and the SPusp45 fused to mature garQ and garI and mature lgnA and lgnI).This work
  pNJFAICmr, pNZ8048c derivative carrying the PCR product E (SPusp45 fused to mature lgnA and lgnI).This work
  pNJFQICmr, pNZ8048c derivative corresponding to the PCR product J (SPusp45 fused to mature garQ and garI).This work
  pNJFQIAICmr, pNZ8048c derivative corresponding to the PCR product K (SPusp45 fused to mature garQ and garI and mature lgnA and lgnI).This work
a SOAT— stab-on-agar test; ADT—agar diffusion test; MPA—microtiter plate assay; Cmr—chloramphenicol resistance. b NIZO—Department of Biophysical Chemistry, NIZO Food Research (Ede, The Netherlands); SD-NUTRYCIAL—Sección Departamental de Nutrición y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid (Madrid, Spain); DPC—Teagasc Dairy Products Research Centre, Moorepark, Fermoy, Co., (Cork, Ireland); CEFAS-BCC— Centre for Environment Fisheries and Aquaculture Science-Bacterial Culture Collection (Suffolk, United Kingdom); LFP-UNIZAR—Laboratory of Fish Pathology, Universidad de Zaragoza (Zaragoza, Spain); Jouy-en-Josas SC—Jouy-en-Josas Strain Collection, INRAE (Jouy-en-Josas, France); CECT—Spanish Type Culture Collection, Universidad de Valencia (Valencia, Spain); RUG-MG—Department of Molecular Genetics, University of Groningen (Haren, The Netherlands).
Table
Table 2. Direct antimicrobial activity and extracellular antimicrobial activity of recombinant L. lactis strains, producing garvicin A (GarA) and/or garvicin Q (GarQ), against different bacterial indicators.
Table 2. Direct antimicrobial activity and extracellular antimicrobial activity of recombinant L. lactis strains, producing garvicin A (GarA) and/or garvicin Q (GarQ), against different bacterial indicators.
StrainSOAT aADT b
L. garvieae CF00021P. damnosus CECT4797L. garvieae CF00021P. damnosus CECT4797
L. lactis subsp. cremoris
  NZ9000 (pMG36c)(-)(-)(-)(-)
  NZ9000 (pJFAI)6.7(-)10.1(-)
  NZ9000 (pJFQI)6.46.010.38.6
  NZ9000 (pJFAIQI)6.65.810.48.9
  NZ9000 (pJFQIAI)6.85.910.28.8
  NZ9000 (pNZ8048c)(-)(-)(-)(-)
  NZ9000 (pNJFAI)8.6(-)11.9(-)
  NZ9000 (pNJFQI)8.37.112.211.2
  NZ9000 (pNJFQIAI)8.16.511.79.5
  WA2-67 (pMG36c)12.620.513.721.7
  WA2-67 (pJFAI)12.820.414.021.8
  WA2-67 (pJFQI)13.621.315.623.2
  WA2-67 (pJFAIQI)12.720.113.821.7
  WA2-67 (pJFQIAI)14.823.016.925.4
  WA2-67 (pNZ8048c)12.320.513.621.3
  WA2-67 (pNJFAI)12.720.113.821.7
  WA2-67 (pNJFQI)12.220.814.022.2
WA2-67 (pNJFQIAI)12.320.313.822.0
L. lactis subsp. lactis
  DPC5598 (pMG36c)7.29.013.720.0
  DPC5598 (pJFAI)7.68.713.519.4
  DPC5598 (pJFQI)7.58.913.619.7
  DPC5598 (pJFAIQI)6.87.612.818.0
  DPC5598 (pJFQIAI)6.26.511.916.7
  DPC5598 (pNZ8048c)7.69.514.020.6
  DPC5598 (pNJFAI)7.89.114.521.3
  DPC5598 (pNJFQI)7.39.414.822.0
  DPC5598 (pNJFQIAI)6.88.612.519.1
  BB24 (pMG36c)7.99.712.418.6
  BB24 (pJFAI)9.910.014.518.3
  BB24 (pJFQI)9.413.414.620.5
  BB24 (pJFAIQI)9.713.814.219.9
  BB24 (pJFQIAI)9.613.914.320.2
  BB24 (pNZ8048c)8.510.113.018.7
  BB24 (pNJFAI)10.313.715.019.3
  BB24 (pNJFQI)10.714.215.120.4
BB24 (pNJFQIAI)10.214.615.020.0
a Direct antimicrobial activity as determined by a stab-on-agar test (SOAT) and b extracellular antimicrobial activity as determined by an agar diffusion test (ADT). Both are expressed as halos of growth inhibition (diameter in mm). Most of the data are means from two independent determinations in triplicate. Control strains are in bold. (-)—no activity.
Table
Table 3. Antimicrobial activity a of supernatants from recombinant L. lactis strains transformed with pMG36c-derived plasmids against virulent L. garvieae strains.
Table 3. Antimicrobial activity a of supernatants from recombinant L. lactis strains transformed with pMG36c-derived plasmids against virulent L. garvieae strains.
StrainL. garvieae
CF00021CF01144CLG4CLG5CLFP28/06JIP29/99
L. lactis subsp. cremoris
  NZ9000 (pMG36c)(-)(-)(-)(-)(-)(-)
  NZ9000 (pJFAI)889076698885
  NZ9000 (pJFQI)9814311210697100
  NZ9000 (pJFAIQI)121169151153149140
  NZ9000 (pJFQIAI)126145101167107108
  WA2-67 (pMG36c)2199103511483911087521
  WA2-67 (pJFAI)2276146919204751566744
  WA2-67 (pJFQI)11,79811,0825935420188644821
  WA2-67 (pJFAIQI)2193174918254681637847
  WA2-67 (pJFQIAI)150,03319,57128,362678033,1149412
L. lactis subsp. lactis
  DPC5598 (pMG36c)1808127118651391042855
  DPC5598 (pJFAI)1529132621331981318960
  DPC5598 (pJFQI)17701338197612912881066
  DPC5598 (pJFAIQI)109610681312921064905
  DPC5598 (pJFQIAI)10336591489811055815
  BB24 (pMG36c)11237904431151479514
  BB24 (pJFAI)23852480161720320361105
  BB24 (pJFQI)25322530172520231151243
  BB24 (pJFAIQI)22382756178620426151459
BB24 (pJFQIAI)25352699164119631661317
a Antimicrobial activity as determined by a microtiter plate assay (MPA) and expressed in bacteriocin units per milligram cell dry weight (BU/mg cdw). Most of the data are means from two independent determinations in triplicate. Control strains are in bold. (-)—no activity.
Table
Table 4. Antimicrobial activity a of supernatants from recombinant L. lactis strains transformed with pNZ8048c-derived plasmids against virulent L. garvieae strains.
Table 4. Antimicrobial activity a of supernatants from recombinant L. lactis strains transformed with pNZ8048c-derived plasmids against virulent L. garvieae strains.
StrainL. garvieae
CF00021CF01144CLG4CLG5CLFP28/06JIP29/99
L. lactis subsp. cremoris
  NZ9000 (pNZ8048c)(-)(-)(-)(-)(-)(-)
  NZ9000 (pNJFAI)11269971440129516001263
  NZ9000 (pNJFQI)121410691395119414401220
  NZ9000 (pNJFQIAI)105111881297116715411255
  WA2-67 (pNZ8048c)16681002897364987369
  WA2-67 (pNJFAI)18539891000412902385
  WA2-67 (pNJFQI)2150135112375651163486
  WA2-67 (pNJFQIAI)2021122311495951245491
L. lactis subsp. lactis
  DPC5598 (pNZ8048c)21231258231719810971096
  DPC5598 (pNJFAI)27602699344331716101770
  DPC5598 (pNJFQI)26051496233426614991492
  DPC5598 (pNJFQIAI)11508801800981480920
  BB24 (pNZ8048c)12036933971581022685
  BB24 (pNJFAI)33983102234033929891597
  BB24 (pNJFQI)37053663273239741721603
BB24 (pNJFQIAI)35023375254129950162001
a Antimicrobial activity as determined by a microtiter plate assay (MPA) and expressed in bacteriocin units per milligram cell dry weight (BU/mg cdw). Most of the data are means from two independent determinations in triplicate. Control strains are in bold. (-)—no activity.
Table
Table 5. Purification of NisZ and GarQ produced by L. lactis subsp. cremoris WA2-67 (pJFQI) and purification of NisZ, GarA and GarQ produced by L. lactis subsp. cremoris WA2-67 (pJFQIAI).
Table 5. Purification of NisZ and GarQ produced by L. lactis subsp. cremoris WA2-67 (pJFQI) and purification of NisZ, GarA and GarQ produced by L. lactis subsp. cremoris WA2-67 (pJFQIAI).
Purification StageVolume
(mL)		Total
A254 a		Total
Antimicrobial Activity
(BU) b		Specific
Antimicrobial Activity (BU/A254) c		Increase in Specific
Antimicrobial Activity (Fold) d		Recovery Antimicrobial Activity
(%)
L. lactissubsp. cremorisWA2-67 (pJFQI)
Culture supernatant100030,50010.6 × 1063471100
Ammonium sulfate precipitation100193012.3 × 106637018116
Gel filtration chromatography1858722.1 × 1062410720
Cation-exchange chromatography507511.3 × 106150,670434107
Hydrophobic-interaction chromatography158.41.0 × 106119,0503439
Reversed-phase chromatography
 Fraction 80.3000.32550.0 × 103153,8504430.5
 Fraction 140.4000.0562.3 × 10341,0701180.02
L. lactissubsp. cremoris WA2-67 (pJFQIAI)
Culture supernatant100028,4001.4 × 10849301100
Ammonium sulfate precipitation10015101.5 × 109993,3802011071
Gel filtration chromatography1858322.3 × 108276,44056164
Cation-exchange chromatography501191.8 × 10915,13031286
Hydrophobic-interaction chromatography1513.816.1 × 1061,166,67023711.5
Reversed-phase chromatography
 Fraction 70.3000.12521,400171,200350.015
 Fraction 90.4500.218220010,10020.0016
 Fraction 120.5000.040200500010.00014
a Absorbance at 254 nm (A254) multiplied by the volume in milliliters. b Antimicrobial activity in bacteriocin units per milliliter (BU/mL), determined by an MPA against L. garvieae CF00021 and multiplied by the total volume in milliliters. c Specific antimicrobial activity expressed as the total antimicrobial activity (BU) divided by the total A254. d Specific antimicrobial activity of a fraction (BU/A254) divided by the specific antimicrobial activity of the initial culture supernatant (BU/A254).
Table
Table 6. Peptide fragments identified by MRM-LC-ESI-MS/MS (QTRAP) in eluted purified fractions from supernatants of L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI).
Table 6. Peptide fragments identified by MRM-LC-ESI-MS/MS (QTRAP) in eluted purified fractions from supernatants of L. lactis subsp. cremoris WA2-67 (pJFQI) and L. lactis subsp. cremoris WA2-67 (pJFQIAI).
StrainFractionPeptide SequencePrecursor
MW		Detected m/zRetention TimeDetected TransitionsMS/MS
Confirmation
L. lactis subsp. cremoris
  WA2-67 (pJFQI)14GarQ
 EYHLMNGANGYLTR1638.76546.92 (+3)20.14+
 VNGKYVYR998.54499.77 (+2)8.74+
  WA2-67 (pJFQIAI)9GarQ
 EYHLMNGANGYLTR1638.76546.92 (+3)21.34+
 VNGKYVYR998.54499.77 (+2)10.44+
12GarA
 GKINQYRPY1138.60569.8 (+3)14.74+
 INQYRPY953.48477.24 (+2)15.65+
Amino acid sequence of GarQ: EYHLMNGANGYLTRVNGKYVYRVTKDPVSAVFGVISNGWGSAGAGFGPQH and GarA: IGGALGNALNGLGTWANMMNGGGFVNQWQVYANKGKINQYR. PY. Lysine (K) and arginine (R) residues for hydrolysis by trypsin, in bold. +, positive confirmation.
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MDPI and ACS Style

Feito, J.; Araújo, C.; Arbulu, S.; Contente, D.; Gómez-Sala, B.; Díaz-Formoso, L.; Muñoz-Atienza, E.; Borrero, J.; Cintas, L.M.; Hernández, P.E. Design of Lactococcus lactis Strains Producing Garvicin A and/or Garvicin Q, Either Alone or Together with Nisin A or Nisin Z and High Antimicrobial Activity against Lactococcus garvieae. Foods 2023, 12, 1063. https://doi.org/10.3390/foods12051063

AMA Style

Feito J, Araújo C, Arbulu S, Contente D, Gómez-Sala B, Díaz-Formoso L, Muñoz-Atienza E, Borrero J, Cintas LM, Hernández PE. Design of Lactococcus lactis Strains Producing Garvicin A and/or Garvicin Q, Either Alone or Together with Nisin A or Nisin Z and High Antimicrobial Activity against Lactococcus garvieae. Foods. 2023; 12(5):1063. https://doi.org/10.3390/foods12051063

Chicago/Turabian Style

Feito, Javier, Carlos Araújo, Sara Arbulu, Diogo Contente, Beatriz Gómez-Sala, Lara Díaz-Formoso, Estefanía Muñoz-Atienza, Juan Borrero, Luis M. Cintas, and Pablo E. Hernández. 2023. "Design of Lactococcus lactis Strains Producing Garvicin A and/or Garvicin Q, Either Alone or Together with Nisin A or Nisin Z and High Antimicrobial Activity against Lactococcus garvieae" Foods 12, no. 5: 1063. https://doi.org/10.3390/foods12051063

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