Garvicin A, a Novel Class IId Bacteriocin from Lactococcus garvieae That Inhibits Septum Formation in L. garvieae Strains

Abstract

Lactococcus garvieae 21881, isolated in a human clinical case, produces a novel class IId bacteriocin, garvicin A (GarA), which is specifically active against other L. garvieae strains, including fish- and bovine-pathogenic isolates. Purification from active supernatants, sequence analyses, and plasmid-curing experiments identified pGL5, one of the five plasmids found in L. garvieae [M. Aguado-Urda et al., PLoS One 7(6):e40119, 2012], as the coding plasmid for the structural gene of GarA (lgnA), its putative immunity protein (lgnI), and the ABC transporter and its accessory protein (lgnC and lgnD). Interestingly, pGL5-cured strains were still resistant to GarA. Other putative bacteriocins encoded by the remaining plasmids were not detected during purification, pointing to GarA as the main inhibitor secreted by L. garvieae 21881. Mode-of-action studies revealed a potent bactericidal activity of GarA. Moreover, transmission microscopy showed that GarA seems to act by inhibiting septum formation in L. garvieae cells. This potent and species-specific inhibition by GarA holds promise for applications in the prevention or treatment of infections caused by pathogenic strains of L. garvieae in both veterinary and clinical settings.

INTRODUCTION

Lactococcus garvieae is one of the most important bacterial pathogens affecting different farmed and wild fish species in both fresh and marine waters, although its major impact is on the trout farm industry (1). To avoid the economic losses of fish lactococcosis, several strategies based on the use of vaccines, probiotics, or bacteriocins have been developed (2–4). However, the host range of L. garvieae is not limited to aquatic species; it has been associated with subclinical mastitis in cows and water buffalos (5, 6) and with pneumonia in pigs (7). L. garvieae has been also isolated from human patients with different clinical conditions, such as urinary tract infections, pneumonia, endocarditis, or septicemia (8, 9). In fact, L. garvieae is currently considered a potential emerging zoonotic pathogen (10). In addition, this microorganism is widespread in the environment, having been isolated from wild birds, rivers, sewage waters, and a wide variety of foodstuffs, including vegetables, meat, sausages, and, particularly, artisan dairy products (11–13).

Recently, the complete genome sequence of L. garvieae 21881, a human clinical strain, has been published (14), and its five plasmids (pGL1 to pGL5) have been characterized at the molecular level (15). This revealed that plasmids pGL1, pGL2, and pGL5 encode putative proteins related to bacteriocin biosynthesis, secretion, and immunity (15). Bacteriocins are ribosomally synthesized antimicrobial peptides of bacterial origin with an antimicrobial spectrum usually directed against species that are closely related or share the same ecological niche. Some bacteriocins have a narrow species range, and their inhibitory spectrum is limited to a few species or to strains of the same species as the producer strain, while other bacteriocins have broad ranges, acting against different bacterial genera (16). The production of these antimicrobial peptides plays an important role in bacterial competition, conferring survival advantages on the producing bacteria (17). Bacteriocins from Gram-positive bacteria are classified into two major classes (16): the lantibiotics (class I; bacteriocins containing posttranslationally modified residues) and the nonlantibiotics (class II; bacteriocins with nonmodified residues except for the formation of disulfide bridges and circular bacteriocins). Class II is divided into four subgroups: classes IIa (pediocin-like bacteriocins), IIb (two-peptide bacteriocins), IIc (cyclic bacteriocins), and IId (linear non-pediocin-like one-peptide bacteriocins). Recently, class IId has been proposed to be subdivided into three groups: (i) sec-dependent bacteriocins, (ii) leaderless bacteriocins, and (c) nonsubgrouped bacteriocins (18).

Lactococcal bacteriocins are represented in all the classes and subclasses described above (19), and some of them have been studied extensively because of their potential applications in the inhibition of spoilage and pathogenic microorganisms in foods. In fact, nisin, produced by some strains of Lactococcus lactis, is one of the best-known bacteriocins and the only one, apart from pediocin PA-1/AcH from Pediococcus acidilactici, that has found commercial application as a food preservative (20, 21). However, only three bacteriocins from L. garvieae have been reported to date: garviecin L1-5 (22), garvicin ML (23), and garvieacin Q (24). This work describes the purification, characterization, and mode of action of garvicin A (GarA), a novel class IId bacteriocin from L. garvieae 21881, which acts exclusively against other L. garvieae strains, including those involved in human, fish, porcine, or bovine disease. Here we show that GarA has potent and species-specific antimicrobial activity; it is active against L. garvieae strains responsible for lactococcosis in fish or mastitis in cows and water buffalos. Thus, GarA holds promise for applications in the prevention or treatment of infections caused by pathogenic strains of L. garvieae in both veterinary and clinical settings.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Lactococcus garvieae 21881 was isolated from the blood of a 74-year-old male patient with septicemia (14). L. garvieae 21881 has been deposited at the “Collection Española de Cultivos Tipo” (CECT) as L. garvieae CECT 8729 under the regulations of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for patent purposes. In addition, we have submitted a patent application (P201300260) for using the garvicin A bacteriocin at the Spanish Patent and Trademark Office of the Ministry of Industry, Tourism, and Trade of Spain.

Enterococcus, Listeria, Streptococcus, and Staphylococcus strains were grown in brain heart infusion (BHI; Oxoid); Carnobacterium, Lactobacillus, Lactococcus, and Pediococcus strains, in de Man, Rogosa, and Sharpe (MRS) medium (Oxoid); and Bordetella, Escherichia, and Salmonella strains, in Luria-Bertani (LB) medium. All strains were incubated at 30°C, without agitation, except for Gram-negative strains, which were grown at 37°C with agitation. Thirty-five isolates of L. garvieae from food (n = 10), diseased fishes (n = 8), diseased ruminants (n = 5), diseased pigs (n = 2), diseased humans (n = 5), and environmental samples (n = 5) were studied as target strains (Table 1). All the species and strains used as indicator microorganisms to determine the spectrum of activity of the bacteriocin are listed in Table 1.

Table 1.

Antimicrobial spectrum of cell-free supernatants from Lactococcus garvieae 21881

Indicator strain	Origin	Sourcea	Inhibition (mm)b at:
			pH 4.8	pH 6.1
Bordetella pertussis NCTC 8616	Human (whooping cough)	FVM	—	—
Carnobacterium piscicola 02/5685	Fish (trout)	FVM	—	—
Enterococcus faecalis TAB 28	Food (cheese)	FVM	—	—
E. faecalis CECT 4039	Food (cheese)	CECT	—	—
Enterococcus faecium 29J	Food (sausage)	FVM	—	—
Lactobacillus acidophilus ATCC 4356T	Human	TNO	—	—
Lactobacillus brevis LB9	Food (wine fermentation)	UV	—	—
Lactobacillus bulgaricus ATCC 11842T	Food (Bulgarian yogurt)	TNO	—	—
Lactobacillus casei ATCC 334	Food (Emmental cheese)	TNO	—	—
Lactobacillus curvatus NCFB 2739T	Food (milk)	TNO	—	—
Lactobacillus delbrueckii CECT 282	Food (meat)	CECT	—	—
Lactobacillus fermentum ATCC 9338	Food	TNO	—	—
L. fermentum LC40	Human (breast milk)	FVM	—	—
Lactobacillus gasseri 2459	Human (vagina)	UO	—	—
Lactobacillus hilgardii LB76	Wine	UV	—	—
Lactobacillus jensenii 2967	Human (vagina)	UO	—	—
Lactobacillus pentosus 128/2	Food (olive fermentation)	CIG	—	—
L. pentosus IG1	Food (olive fermentation)	CIG	—	—
Lactobacillus plantarum NC8	Grass silage	Matforsk	—	—
L. plantarum WCFS1	Human (saliva)	Matforsk	—	—
Lactobacillus reuteri DSM 20016T	Human (intestine)	TNO	—	—
Lactobacillus rhamnosus G32.8	Food (olive fermentation)	CIG	—	—
Lactobacillus sakei 148	Food (sausage)	FVM	—	—
L. sakei NCFB 2714T	Starter of sake (moto)	TNO	—	—
Lactobacillus salivarius HN6	Human (breast milk)	FVM	—	—
Lactococcus garvieae H14	Bird	VISAVET	20	21
L. garvieae Imau 50094	Beetle	FVM	18	17
L. garvieae Pw 1537	Beetle	FVM	24	20
L. garvieae 1205	Buffalo (mastitis)	UFRJ	16	16
L. garvieae CECT 4531T	Cow	CECT	20	20
L. garvieae G-34	Cow (mastitis)	VISAVET	25	20
L. garvieae MAM 75	Cow (mastitis)	VISAVET	21	20
L. garvieae MAM 77	Cow (mastitis)	VISAVET	17	17
L. garvieae CP-1	Fish (trout)	FVM	20	18
L. garvieae BA063090	Fish (eel)	FVM	15	15
L. garvieae 4876/006	Fish (eel)	VISAVET	20	20
L. garvieae 1686	Fish (trout)	VISAVET	15	14
L. garvieae 5457	Fish (trout)	VISAVET	30	25
L. garvieae 5657	Fish (trout)	VISAVET	15	14
L. garvieae 8831	Fish (trout)	VISAVET	21	22
L. garvieae LG-2	Fish (Seriola)	HUJ	18	18
L. garvieae 1204	Food (Salers cheese)	INRA	18	18
L. garvieae 3AA7	Food (Cabrales cheese)	IPLA	19	18
L. garvieae 4AB5	Food (Cabrales cheese)	IPLA	16	15
L. garvieae CAS-2	Food (Casín cheese)	IPLA	15	16
L. garvieae DK 2-25	Food (kajmak)	IMGGE	13	15
L. garvieae N201	Food (Saint-Nectaire cheese)	IPLA	16	16
L. garvieae T1-1	Food (Casín cheese)	IPLA	16	16
L. garvieae T2-17	Food	IPLA	16	16
L. garvieae NRTC0611	Food (vegetable)	HUJ	14	14
L. garvieae Lg80	Food (Morcilla de Burgos)	UB	15	14
L. garvieae 240-88	Human (urine)	IFR	20	20
L. garvieae 306/79	Human (urine)	IFR	14	13
L. garvieae 364-88	Human (blood)	IFR	19	19
L. garvieae BM06/00349	Human (blood)	HRV	15	14
L. garvieae 1481/03	Pig (pleuritis)	VISAVET	20	20
L. garvieae 2497/03	Pig (pericarditis)	VISAVET	20	20
L. garvieae BA06/021331	Water	VISAVET	15	15
L. garvieae DP1	Water	VISAVET	19	17
Lactococcus lactis ATCC 11454	Food (cheese)	ATCC	—	—
L. lactis ESI 153	Food (cheese)	FVM	—	—
Lactococcus lactis subsp. cremoris MG1363	Food	CIT	—	—
L. lactis MG 1614	Food (cheese)	CIT	—	—
Lactococcus lactis subsp. lactis CECT 185T	Food	CECT	—	—
L. lactis subsp. lactis IL1403	Food (cheese)	INRA	—	—
L. lactis subsp. lactis 1585-85	Human (blood)	IFR	—	—
Listeria monocytogenes 51112	Food	FVM	—	—
L. monocytogenes Scott A	Food	FVM	—	—
L. monocytogenes CECT 935 (4b)	Human/animal/food	CECT	—	—
L. monocytogenes CECT 935 (4c)	Human/animal/food	CECT	—	—
Pediococcus acidilactici CECT 98	Food	CECT	—	—
Pediococcus pentosaceus FBB63	Vegetable fermentation	TNO	—	—
Salmonella strain S79	Feces	FVM	—	—
Staphylococcus aureus CECT 86T	Bovine (clinical)	CECT	—	—
Staphylococcus epidermidis CECT 231	Human (nose)	CECT	—	—
Streptococcus bovis DSM 20480T	Cow	DSMZ	—	—
Streptococcus dysgalactiae DSM 20662T	Cow	DSMZ	—	—
Streptococcus lactarius DSM 23027T	Human (breast milk)	DSMZ	—	—
Streptococcus mitis DSM 12643T	Human (oral cavity)	DSMZ	—	—
Streptococcus oralis CECT 907T	Human	CECT	—	—
Streptococcus parasanguinis DSM 6778T	Human (throat)	DSMZ	—	—
Streptococcus peroris DSM 12393T	Human (oral cavity)	DSMZ	—	—
Streptococcus pyogenes CECT 985T	Human	CECT	—	—
Streptococcus pneumoniae DSM 20566T	Human	DSMZ	—	—
Streptococcus salivarius CECT 805T	Human (rheumatism)	CECT	—	—
Streptococcus thermophilus ST20	Starter culture (yogurt)	TNO	—	—
Streptococcus uberis CECT 994T	Bovine mastitis	CECT	—	—

^aATCC, American Type Culture Collection (Manassas, VA); CECT, Colección Española de Cultivos Tipo (Universidad de Valencia, Burjasot, Spain); CIG, Colección Instituto de la Grasa (Seville, Spain); CIT, Cranfield Institute of Technology (United Kingdom); DMSZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany); FVM, Facultad de Veterinaria, Universidad Complutense (Madrid, Spain); INRA, Institut National de la Recherche Agronomique (Jouy-en-Josas, France); UO, Universidad de Oviedo (Spain); IPLA, Instituto de Productos Lácteos de Asturias (Villaviciosa, Spain); Matforsk (Ås, Norway); TNO, Nutrition and Food Research (Zeist, The Netherlands); UV, Universidad de Valencia (Burjasot, Spain), UB, Universidad de Burgos (Spain); HUJ, Hiroshima University Japan (Japan); UFRJ, Universidade Federal do Rio de Janeiro (Brazil); IMGGE, Institute of Molecular Genetics and Genetic Engineering (Belgrade, Serbia); HRV, Hospital Royo Villanova (Zaragoza, Spain); IFR, Institute of Food Research (Norwich, United Kingdom); VISAVET, Centro de Vigilancia Sanitaria (Madrid, Spain).

^bDiameter of the halo of inhibition. -, no inhibition. Antimicrobial activity was determined by the agar diffusion test with bacteriocin-active cell-free supernatants of L. garvieae 21881 at the pH values indicated.

Bacteriocin assays.

Antimicrobial activity was assayed by the agar diffusion test using the strains listed in Table 1 as indicator strains. Briefly, the sterilized MRS medium, which contained 1.2% agar, was held at 50°C before being inoculated with ca. 10⁵ cells of the indicator strain and then poured onto petri dishes. Once the mixture was solidified, wells (diameter, 4.0 mm) were made with a sterile cork borer. Each well was filled with 50 μl of a filter-sterilized (0.22 μm; Millipore) cell-free supernatant (CFS) from a 16-h broth culture of L. garvieae 21881 (pH ∼4.9) or with the same CFS adjusted to pH ∼6.15 with 1 N NaOH. After 1 h at 4°C, plates were incubated at 30°C for 16 h, and then the diameters of the inhibition zones were recorded.

Bac⁺ CFSs were either heated at 100°C for 5 min, autoclaved at 121°C for 15 min, or treated with 0.1 mg/ml of proteinase K. The antimicrobial activities after and before the treatments were quantified by a microtiter plate assay (25) using L. garvieae Lg80 and L. garvieae 8831 as the indicator strains. During the purification process, GarA activity was quantified by this microtiter plate assay, using L. garvieae Lg80, L. garvieae 8831, and L. garvieae 3AA7 as the indicator strains. In addition, the inhibitory activity of purified GarA was quantified by using all the strains listed in Table 1 as indicators.

To check for bacteriocin production by novobiocin-treated L. garvieae 21881 cultures on solid medium, a “spot-on-lawn” method was used. Briefly, a 5-μl drop of a broth culture of a novobiocin-treated L. garvieae 21881 isolate was placed on the surface of each MRS agar plate and was incubated at 30°C for 4 h. Then the plates were overlaid with 4.5 ml soft agar (MRS medium plus 0.75% [wt/vol] agar) inoculated with ca. 10⁵ CFU/ml of the indicator strain L. garvieae Lg80 or L. garvieae 3AA7. Plates were further incubated at 30°C for 16 to 18 h and were examined for clear halos of inhibition around the spots. Spots of nontreated L. garvieae 21881 were also assayed as positive controls.

Purification of garvicin A.

All the GarA purification steps were carried out at room temperature, and all of the chromatographic equipment and media were purchased from Amersham Biosciences Europe GmbH (Freiburg, Germany). The bacteriocin was purified by using the supernatant of a 5-liter culture of L. garvieae 21881 grown for 24 h in MRS broth (Biokar Diagnostics) at 30°C (optical density at 600 nm [OD₆₀₀], 3.0) and following the protocol of Cintas et al. (26). Briefly, the cells were removed by centrifugation at 10,000 × g for 10 min at 4°C, and the CFS was mixed with 20 g per liter of Amberlite XAD-16 (Sigma-Aldrich, St. Louis, MO) by stirring at room temperature for 2 h. The matrix was washed with 250 ml of distilled water and 187.5 ml of 40% (vol/vol) ethanol in distilled water, and the bacteriocin activity was eluted with 500 ml of 70% (vol/vol) 2-propanol in distilled water (pH 2.0). All washing and elution steps were carried out by decanting. The eluate was further subjected to cation-exchange (SP Sepharose Fast Flow) and hydrophobic-interaction (Octyl-Sepharose CL-4B) chromatography as described previously (27). The eluted fractions showing bacteriocin activity (Bac⁺) were subjected to reverse-phase chromatography (RPC) in a 3-ml Resource RPC column (GE Healthcare) coupled to a fast protein liquid chromatography (FPLC) system. The bacteriocin was eluted from the RPC column with a linear gradient of 2-propanol (Merck) in aqueous 0.1% (vol/vol) trifluoroacetic acid. Fractions showing inhibitory activity after RPC-FPLC were pooled and rechromatographed to obtain pure bacteriocin. The purity of bacteriocin fractions was checked by SDS-PAGE as described below.

SDS-PAGE.

During the purification process, the RPC-FPLC-eluted fractions of GarA were analyzed in duplicate by Tris-Tricine SDS-PAGE using an 18% acrylamide resolving gel (28). After electrophoresis at 100 mV for 2 h, one gel was silver stained, while the other was used to detect the inhibitory activity in an overlay assay as described previously (27). L. garvieae Lg80 was used as the indicator strain. Precision Plus Protein Dual Xtra standards (Bio-Rad) were used as molecular weight standards.

Mass spectrometry analysis.

The molecular weight of purified garvicin A was determined by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), using 2,5-dihydroxybenzoic acid as the matrix, at the Institute of Plant Biochemistry and Photosynthesis (IBVF-CSIC-US, Seville, Spain). Purified garvicin A and a tryptic digest of this bacteriocin were analyzed by MALDI-TOF MS to identify peptide masses before use for tandem mass spectrometry (MS-MS). MS-MS was used to obtain de novo amino acid sequences of the selected peptide fragments. Both MS and MS-MS were performed in a MALDI-TOF/TOF mass spectrometer from Applied Biosystems (4700 Proteomics Analyzer), using alpha-cyano-4-hydroxycinnamic acid as the matrix, at the Proteomic Service Facility of the Genomic and Proteomic Center (Facultad de Farmacia, UCM, Madrid, Spain).

Mode of action.

To study the effect of the bacteriocin on sensitive cells, L. garvieae 8831 was treated with a Bac⁺ CFS from L. garvieae 21881. For this purpose, 2 ml of a filter-sterilized CFS from L. garvieae 21881 (10,240 bacteriocin units [BU] ml⁻¹) was added to 2-ml cultures of L. garvieae 8831 at different growth stages (OD₆₀₀, 0.25, 0.56, 0.95, and 1.10), and the cultures were further incubated at 30°C. At intervals of 5, 15, 30, 60, and 120 min, aliquots were removed to determine the number of viable cells.

Electron microscopy.

Samples were taken from exponentially growing cultures (OD₆₀₀, 0.63) of L. garvieae 8831 before and after treatment (up to 180 min) with a filter-sterilized CFS from L. garvieae 21881 (10,240 BU ml⁻¹). Cells were harvested by centrifugation at 5,900 × g for 10 min and were washed twice in phosphate buffer (0.1 M potassium phosphate buffer, pH 7.4). Samples were fixed in 2.5% (vol/vol) glutaraldehyde (Merck) in phosphate buffer for 12 h, washed with the same buffer, and postfixed in 1% osmium tetroxide plus 3% potassium ferrocyanide for 30 min at room temperature. After fixation, the samples were rinsed three times with distilled water, gradually dehydrated with an acetone gradient (30, 50, 70, 80, 90, 95, and 100%), embedded gradually in epoxy resin (Spurr), and processed in an Ultracut E microtome (Reichert Jung). Thin sections were visualized with a JEOL 1010 electron microscope at 100 kV at the Centro Nacional de Microscopía, UCM (Madrid, Spain).

Plasmid extraction and curing.

Plasmid extraction and analysis were carried out according to the method described by Anderson and McKay (29). As a reference, the parental strain L. garvieae 21881, containing pGL1 (4,536 bp; accession no. NC_016969), pGL2 (4,572 bp; NC_016981), pGL3 (12,948 bp; NC_016970), pGL4 (14,006 bp; NC_016971), and pGL5 (68,798 bp; NC_016982), was used (15). Plasmids were cured as described previously (30) with some modifications. An 18-h MRS broth culture of L. garvieae 21881 (OD₆₀₀, 1.14; ca. 2.2 × 10⁸ CFU/ml) was inoculated into 10 ml MRS broth (final concentration, ca. 10² CFU/ml) containing increasing concentrations of novobiocin (Sigma-Aldrich), ranging from 0.125 to 8 μg/ml, and was incubated at 30°C for 24 h. After incubation, the culture containing the highest concentration of the curing agent that still allowed visible growth was selected, and appropriate dilutions were plated on MRS agar. After incubation at 30°C, isolated colonies were randomly selected for further plasmid analysis and bacteriocin production studies in both solid and broth media, as described above. As a control, MRS broth without novobiocin was inoculated with L. garvieae 21881 and was processed in the same manner.

PCRs.

To check for the presence of the structural genes for garvicin A (lgnA) and its putative immunity protein LgnC (lgnC), the primer pairs lgnA-F (5′-ATTTAATACGGACGGTATTGAT-3′)/lgnA-R (5′-GGAGTAAAAAGATGGAAAACAA-3′) and LGAI-F (5′-AGAAAATGGGCTAACTCCGG-3′)/LGAI-R (5′-ATGAATAAAACAGAAATAATGACT-3′) were designed. Amplification was carried out in 100-μl reaction mixtures containing 2.5 mM MgCl₂, 1× reaction buffer, 100 μM each deoxynucleoside triphosphate (dNTP), 100 pmol of each primer, 5 U of Taq DNA polymerase (BioTools), and 250 ng of DNA. DNAs from L. garvieae 21881 and its derivative L. garvieae 21881-N1 (cured of plasmid pGL5; not a bacteriocin producer) were used as the templates. Amplification included denaturation at 92°C for 2 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 47°C for 1 min 30 s, and polymerization at 72°C for 2 min, and a final extension of 72°C for 10 min, using a GeneAmp PCR system 2400 thermal cycler (Perkin-Elmer Corporation, Norwalk, CT).

DNA and amino acid sequence analysis.

Searches for DNA and amino acid similarities in nucleotide and protein databases were carried out using the Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/) (31). Searches for promoter sequences were carried out with the Neural Network Promoter Prediction Web interface (http://www.fruitfly.org/seq_tools/promoter.html) (32). For the detection of Rho-independent terminators, the ARNold Web server (http://rna.igmors.u-psud.fr/toolbox/arnold/) was used (33). For alignment of the amino acid sequences of the leader and mature peptides of the bacteriocins, the ClustalW2 multiple sequence alignment program was used (34) (http://www.ebi.ac.uk/Tools/msa/clustalw2/). For physicochemical analysis of peptides (isoelectric point, molecular weight), the WinPep program was used (35) (http://n.ethz.ch/∼lhennig/winpep.html).

RESULTS

Antimicrobial spectrum and preliminary characterization.

The cell-free supernatants (CFSs) from L. garvieae 21881 showed a narrow inhibitory spectrum: they were active only against other strains of the same species (Table 1). All L. garvieae strains used as indicators (n = 35) were very sensitive to the bacteriocin-containing (Bac⁺) CFSs. The degree of sensitivity was strain dependent and was not related to the origin of the strain. The inhibitory activities of crude (pH ∼4.9) and pH-adjusted (pH ∼6.15) CFSs were similar. Bac⁺ CFSs retained 100% of their original activities against L. garvieae Lg80 (20,480 BU ml⁻¹) and L. garvieae 3AA7 (10,240 BU ml⁻¹) after heating at 100°C for 5 min and 25% of their activities (5,120 BU ml⁻¹ and 2,560 BU ml⁻¹, respectively) after autoclaving (121°C, 15 min). In contrast, the antimicrobial compound was sensitive to proteinase K.

Moreover, the CFS was also active (100% of its antimicrobial activity) when the indicator strain was incubated at 18°C, a temperature usually associated with trout lactococcosis outbreaks (1).

Purification of garvicin A.

GarA was isolated from the supernatant of a 5-liter broth culture of L. garvieae 21881. The behavior of GarA throughout the purification process was that of a cationic and hydrophobic substance. Two RPC-FPLC runs were necessary to obtain fractions containing pure bacteriocin (data not shown). SDS-PAGE analysis showed a single peptide band with an apparent molecular size of 5 kDa (Fig. 1A), which showed inhibitory activity against L. garvieae Lg80 in the corresponding SDS-PAGE activity gel (Fig. 1B). The inhibitory spectrum of purified GarA was identical to that of a Bac⁺ CFS of L. garvieae 21881 (not shown): it was active only against other L. garvieae strains. The inhibitory activity of pure GarA had titers of 25,600, 12,800, and 12,800 BU ml⁻¹ against L. garvieae Lg80, L. garvieae 3AA7, and L. garvieae 8831, respectively.

Fig 1.

MALDI-TOF mass spectra of purified garvicin A (GarA). [M + H]⁺, monoisotopic peak of GarA; [M + 2H]²⁺, doubly charged ion of GarA. a.u., absorbance units. (Inset) SDS-PAGE (A) and bioassay (B) of purified GarA. L. garvieae Lg80 was used as the indicator strain. MWM, molecular weight marker (in thousands).

Mass spectrometry and DNA and protein sequence analyses of garvicin A.

MALDI-TOF MS analysis of purified GarA (using 2,5-dihydroxybenzoic acid as the matrix) indicated that a monoisotopic peak ([M + H]⁺) of the bacteriocin was present, suggesting that the molecular mass of GarA is 4,678.5 Da (Fig. 1). In addition, a peak of 2,338.5 Da, corresponding to the doubly charged ions ([M + 2H]²⁺) of GarA, was detected (Fig. 1). However, MALDI-TOF MS of GarA in reflector mode using alpha-cyano-4-hydroxycinnamic acid as the matrix resulted in the fragmentation of the peptide, generating one main fragment of 1,841.96 Da and three peaks of 1,451.73, 1,713.86, and 2,090.01 Da, respectively (not shown). After digestion with trypsin, a main peptide of 953.48 Da and a peptide of 1,841.95 Da were obtained. The amino acid sequences obtained by de novo MS-MS peptide mapping of all of the peptides mentioned above are shown in Fig. 2. All peptide fragments were 100% identical to the deduced carboxyl end of the putative bacteriocin encoded by orf-37 of L. garvieae 21881 plasmid pGL5 (15) (Fig. 2). orf-37 encodes a peptide of 63 amino acids (aa), with a typical double-glycine leader peptide that, once processed, results in a 43-aa mature peptide with a theoretical molecular mass of 4,645.25 Da and an isoelectric point of 10.128 (Fig. 2). The experimentally determined molecular mass of GarA (4,678.59 Da) differed by 33 Da from that deduced from orf-37, a fact that could be due to the oxidization of two methionine residues.

Fig 2.

Amino acid sequence of garvicin A (GarA) deduced from the DNA sequence of orf-37 in plasmid pGL5 of L. garvieae 21881 (15) (a) and comparison with the amino acid sequences obtained by de novo MS-MS peptide mapping of purified GarA (b to f). (a) The double-glycine leader peptide of GarA is shown in boldface. The arrow indicates the Gly-Gly cleavage site of the peptide. The theoretical mass of the mature bacteriocin is given on the right. (b, c, d, and e) Peptides obtained by physical fragmentation of GarA after MALDI-TOF MS. (f) Peptide obtained after digestion with trypsin. The asterisk indicates the trypsin cleavage site after lysine (K). A nontryptic peptide of 1,841.95 Da with the same amino acid sequence as the 1,841.96-Da peptide (c) was also obtained. Molecular masses are given on the right.

The mature GarA peptide showed no significant homology with any of the bacteriocins described previously. However, the unprocessed prebacteriocin GarA was similar to a putative bacteriocin encoded by orf-1 in L. garvieae 21881 plasmid pGL2 (Table 2). The leader peptides of GarA and garvieacin Q from L. garvieae BCC 43578 were 95% identical, but there were almost no similarities within the respective mature bacteriocins (Fig. 3). Similarly, the leader peptide of GarA was very similar to the double-glycine leader peptides of other lactococcins belonging to the class IId bacteriocins, such as lactococcin A (36) and lactococcin B (37), produced by L. lactis strains (Fig. 3).

Table 2.

Proteins encoded by the garvicin A locus of L. garvieae 21881 and their closest homologues

L. garvieae 21881 protein (no. of aa)	L. garvieae 21881 locus tag	Homologue
		Protein (no. of aa)	Locus tag	% Id/% Cova	Comments	GenBank accession no.
LgnAb (63)	PGL5p51	ORF-1 (71)	PGL2_p3	50/100	Bacteriocin-like peptide (L. garvieae 21881)	YP_005352349
		GarQ (70)		95/31	Prepeptide garvieacin Q (L. garvieae BCC 43578)	JN605800
LgnI (88)	PGL5p50	—c (87)	LBPG_01707	38/80	Putative enterocin A immunity protein (L. paracasei subsp. paracasei 8700:2)	ZP_04674005
LgnC (715)	PGL5p49	LcnC (715)	L82520	74/99	Lactococcin A ABC transporter ATP binding and permease (L. lactis subsp. lactis IL1403)	NP_266235
		ORF-4 (>388)d		94/54	Putative ABC-type bacteriocin transporter protein, partial (L. garvieae BCC 43578)	JN605800
LgnD (475)	PGL5p48	LcnD (473)	L84721	58/99	Lactococcin A ABC transporter permease (L. lactis subsp. lactis IL1403)	NP_266236

^aId, identity; Cov, coverage.

^bPreviously annotated as ORF-37 (1).

^cUnnamed.

^dPartial ORF.

Fig 3.

Alignments of the amino acid sequences of double-glycine leader peptides (A) and mature peptides (B) of garvicin A and other, similar class IId bacteriocins. The sequences were aligned with the ClustalW2 software at the EMBL-Ebi online server. Asterisks, dots, and double dots indicate fully, strongly, and weakly conserved residues, respectively. The accession numbers are YP_005352403 for garvicin A (15), AEN79392 for garvieacin Q (24), M63675 for lactococcin A (36), and S38128 for lactococcin B (37).

Analysis of pGL5 revealed that just downstream of orf-37 (now renamed lgnA), there were three additional genes potentially related to GarA biosynthesis (Fig. 4A). The first one (lgnI) encodes a putative 88-aa protein (pI 9.72) showing 38% identity with the putative immunity protein of enterocin A in Lactobacillus paracasei subsp. paracasei 8700:2 (ZP_04674005). A putative promoter sequence containing the typical −10 and −35 regions was found upstream of lgnA, while two inverted repeats of 8 bp (separated by 4 bp), which may function as a Rho-independent transcription terminator, were found just downstream of lgnI. This organization suggests that the lgnA and lgnI genes are expressed on the same transcript (Fig. 4B).

Fig 4.

Schematic representation of the locus for garvicin A (GarA) production (A) and detailed analysis of DNA sequences of putative promoters and Rho-independent terminators (B). (A) P1 and P2 are putative promoter sequences, and T1 and T2 are putative Rho-independent transcription terminators. The genes encoding garvicin A (lgnA; previously designated orf-37 [15]) and its immunity protein (lgnI) and those encoding the ABC transporter (lgnC) and the accessory protein (lgnD) seem to form two independent transcriptional units. (B) The putative promoters P1 and P2 were detected with the Neural Network Promoter Prediction online server (32), with a promoter score cutoff of 0.9. The typical −35 and −10 boxes and the ribosome binding sites (RBS) are shown; +1 indicates the putative transcription start. “Met” in the P1 and P2 sequences indicates the methionine residues of GarA and LgnC, respectively. “Stop” in the P2 sequence indicates the termination codon of lgnI. Note that promoter P2 and terminator T1 (sequence underlined in P2) overlap. The Web tool ARNold (33) was used to predict Rho-independent terminators; base pairs of the hairpin are shown in boldface and apical loops in italics. The predicted free energy of terminator hairpins (kcal/mol) is given in parentheses.

Downstream of lgnI, lgnC (Fig. 4A) encodes a 715-aa putative protein with 74% and 95% identity with the ABC transporters of lactococcin A and garvieacin Q, encoded by L. lactis IL1403 lcnC and L. garvieae BCC 43578 orf-4, respectively (Table 2). Just 10 bp downstream of lgnC, lgnD encodes a putative 475-aa protein with 58% identity to the ABC transporter permease of lactococcin A (Table 2). Interestingly, a putative promoter sequence was found upstream of lgnC, just overlapping the Rho-independent transcription terminator behind lgnI (Fig. 4B). In addition, two inverted repeats of 8 bp (separated by 7 bp), which may function as a Rho-independent transcription terminator, were found downstream of lgnD, indicating cotranscription of lgnC and lgnD (Fig. 4B).

Plasmid curing.

L. garvieae 21881 harbors five plasmids, pGL1 (4.53 kb), pGL2 (4.57 kb), pGL3 (12.94 kb), pGL4 (14.00 kb), and pGL5 (68.79 kb), that have been sequenced recently (15). Treatment of this L. garvieae strain with novobiocin (1 μg/ml) led to a mix of lactococcal cells containing all the possible plasmid combinations, from none to five (results not shown). However, only those strains that had lost plasmid pGL5 became bacteriocin nonproducers (Bac⁻). The plasmid profile of one of these Bac⁻ strains, L. garvieae 21881-N1, is shown in Fig. 5. Surprisingly, these Bac⁻ isolates were still resistant to GarA (data not shown).

Fig 5.

Agarose gel electrophoresis of plasmid DNA from the wild-type bacteriocin producer L. garvieae 21881 (Bac⁺) and L. garvieae 21881-N1, a novobiocin-treated non-bacteriocin-producing strain (Bac⁻). The sizes of the plasmids are as follows: pGL1, 4.53 kb; pGL2, 4.57 kb; pGL3, 12.94 kb; pGL4, 14.00 kb; pGL5, 68.79 kb. Chr, chromosomal DNA.

Two DNA fragments of 205 and 261 bp were amplified with the primer pairs lgnA-F/lgnA-R and LGAI-F/LGAI-R, respectively, using DNA from the wild-type strain L. garvieae 21881. However, no amplification was obtained from L. garvieae 21881-N1, indicating that the lgnA and lgnI genes were lost together with plasmid pGL5.

Bactericidal action.

The addition of a Bac⁺ CFS of L. garvieae 21881 to MRS broth cultures of the trout pathogen L. garvieae 8831 resulted in rapid killing of these sensitive cells. After just 5 min of exposure, the viability of the cultures declined 5 to 6 log CFU per ml (Fig. 6). This decrease was even more pronounced when the bacteriocin was added to exponentially growing cultures (OD₆₀₀, 0.56): the number of viable cells per ml decreased from 1.8 × 10⁸ to 1.0 × 10² after 5 min of incubation (Fig. 6), a result that indicated a bactericidal mode of action.

Fig 6.

Bactericidal effect of crude garvicin A on the sensitive strain L. garvieae 8831 at different phases of growth. The bacteriocin was added to broth cultures of L. garvieae 8831 at an OD₆₀₀ of 0.25 (●), 0.56 (■), 0.95 (△), or 1.10 (◇). Samples were withdrawn at different time intervals, and cell viability was calculated.

Transmission electron microscopy.

Exponentially growing cultures of L. garvieae 8831 were visualized by electron microscopy before and after treatment with an active CFS from L. garvieae 21881. In control cultures, short chains of coccoid cells were observed (Fig. 7A), while after treatment with the CFS, L. garvieae 8831 cells lost their typical spherical shape and became elongated, with irregular, rod-like forms (Fig. 7B and C). These elongated cells showed constriction at their equatorial zones, where the septum primordia were being synthesized. However, after treatment with the bacteriocin, the formation of the septa was inhibited, since none of the cells observed was able to form a completed cross-wall.

Fig 7.

Effect of garvicin A on the morphology of exponentially growing cells of L. garvieae 8831 as observed by electron microscopy. (A) Untreated control cultures; (B and C) cultures treated with the bacteriocin (10,240 BU ml⁻¹) for 30 min. Arrows indicate the septum primordia. The bracket in panel C indicates the observed breakage in the cell wall.

DISCUSSION

In this work, the purification and characterization of GarA, a novel bacteriocin produced by L. garvieae 21881, are described. This bacteriocin is synthesized as a prepeptide containing a typical double-glycine leader peptide, which, once processed, results in mature, active GarA. The mature GarA peptide showed no homology with any previously known bacteriocin, although its leader peptide was nearly identical to that of garvieacin Q (Fig. 3) (24). This fact could explain the high level of similarity between the ABC transporters LgnC of L. garvieae 21881 and ORF-4 of L. garvieae BCC 43578, involved in the secretion and processing of the bacteriocins GarA and GarQ, respectively (Table 2). On the basis of the genetic organization of the genes involved in GarA biosynthesis and the amino acid sequence of GarA, which lacks the typical pediocin-like motif YGNGVXC and has no modified amino acids, this bacteriocin was considered to belong to the class IId one-peptide bacteriocins (16). The identification of promoter and Rho-independent terminator sequences indicated that, most probably, lgnAI and lgnCD form two independent operons. Interestingly, the intercistronic region between lgnI and lgnC contains two overlapping transcription signals: the lgnI terminator (T1) and the lgnC promoter (P2). The −10 region of P2 is contained within the T1 terminator. This overlapping of the two transcription signals could serve regulatory purposes, as has been reported previously (38).

The genes responsible for GarA production, immunity, and secretion have been located in plasmid pGL5 of L. garvieae 21881, which has been sequenced recently (15). In addition, analysis of the DNA sequences of L. garvieae 21881 plasmids pGL1 and pGL2 revealed the presence of two putative bacteriocins and an immunity protein. In pGL1, orf-3 encodes a 71-aa putative peptide with a double-glycine prepeptide and with 42.9% identity to GarA. The mature peptide would have 51 aa, a theoretical mass of 5,247.89 Da, an isoelectric point of 8.0, and 19.6% identity with mature GarA. In pGL2, orf-1 also encodes a 71-aa putative peptide with 50% identity to GarA. This putative bacteriocin presents a double-glycine prepeptide with 65% identity with the GarA prepeptide. The mature peptide would have 51 aa, a theoretical mass of 5,118.65 Da, and an isoelectric point of 9.6. This putative bacteriocin showed 42.3% identity with GarA. Immediately downstream of orf-1, a gene (entI) encoding a putative 97-aa immunity protein with a pI of 9.60 was found. It showed 41% identity with a hypothetical protein of L. lactis MG1363, belonging to the enterocin I immunity family (Pfam08951), and 25.8% identity with LgnI, the putative immunity protein for GarA.

None of the putative bacteriocin peptides encoded by orf-1 in pGL1 or orf-3 in pGL2 were detected in the CFS during the purification process or in the mass spectrometry analysis. Thus, the ABC transporter and accessory protein encoded on pGL5 (lgnC and lgnD) could be specific for GarA and unable to promote the secretion and processing of the other putative bacteriocins (ORF-1 and ORF-3) that, based on the data obtained in the array experiments (data not shown), are expressed by L. garvieae 21881. Additionally, curing plasmid pGL5 resulted in the loss of GarA production, thus indicating that this was the only bacteriocin secreted into the medium by L. garvieae 21881. L. garvieae strain 21881-N1, lacking pGL5, was not sensitive to GarA, suggesting that other mechanisms, apart from the immunity protein LgnI, must be involved in the resistance of L. garvieae 21881 to GarA. Interestingly, immunity or resistance to GarA did not depend on any plasmid-encoded factor, since a novobiocin-derived L. garvieae 21881 strain lacking the five plasmids (pGL1, pGL2, pGL3, pGL4, and pGL5) was still resistant to GarA (results not shown). Previously, the existence of more than one resistance mechanism to lactococcal bacteriocins has been described. For example, immunity to class I bacteriocins targeting lipid II, such as nisin, depends on two separate mechanisms that work together to confer immunity on the producer cells: an ABC transporter system (39) and a dedicated immunity protein (LanI) (40).

To date, only three bacteriocins produced by L. garvieae strains have been reported: garviecin L1-5 (22), which has not been described to a molecular level yet; garvieacin Q (GarQ) (24); and garvicin ML (GarML) (23). In contrast to GarA, garviecin L1-5, GarQ, and GarML display broad inhibitory spectra; they are active not only against L. garvieae strains but also against other Gram-positive species, such as Enterococcus faecium, Listeria monocytogenes, Listeria ivanovii, Pediococcus pentosaceus, Pediococcus acidilactici, Lactobacillus casei, Lactobacillus sakei, Lactobacillus plantarum, Propionibacterium spp., Clostridium spp., Streptococcus pneumoniae, and L. lactis (22–24). However, there are other lactococcal bacteriocins, produced by different strains of L. lactis, with narrow activity spectra, restricted mainly to other L. lactis strains. These include lactococcin G (41), lactococcin Q (42), lactococcin A (36), lactococcin B (43, 44), lactococcin M (43, 44), lactococcin 972 (45), and LsbA and LsbB (46).

The CFS from L. garvieae 21881 exhibited a potent bactericidal effect on the indicator strain L. garvieae 8831, isolated from rainbow trout with lactococcosis (47). Five minutes after the addition of a Bac⁺ CFS to L. garvieae 8831 cultures, nearly 100% of the population died. This bactericidal effect was even more drastic when L. garvieae 8831 cultures were in the exponential phase of growth. The fact that this is the moment at which cells are dividing most actively suggested that the mechanism of action of this bacteriocin was based on inhibition of cell division. This hypothesis was reinforced by the results of electron microscopy, which showed that after the CFS treatment, cells became elongated, did not divide, and, finally, were lysed. Therefore, the GarA target seems to be the inhibition of cell wall biosynthesis, most probably by inhibiting septum formation, as described previously for lactococcin 972 (48). The processes of cell elongation and septum formation are linked in Gram-positive cocci, taking place at an annular band located in the equatorial zone of the cell. Similarly to L. garvieae, lactococcin 972-treated L. lactis cells presented only a primordial septum and suffered an elongation that preceded the arrest of macromolecular synthesis and the death of the cell (48). This suggests that both lactococcin 972 and GarA inhibit septum invagination rather than septum initiation.

The lantibiotic nisin and other lantibiotics kill bacteria primarily by forming pores together with the essential cell wall-biosynthetic molecule lipid II but can also act by sequestering lipid II from the sites where bacterial cell wall synthesis occurs, thus inhibiting septum formation (49, 50). Recently, Lcn972 has been described as the first nonlantibiotic bacteriocin that specifically interacts with the cell wall precursor lipid II (51). Interestingly, some bacteriocins may interfere with septum formation when used at concentrations much higher than those required for their membrane-targeted pore-forming action. Thus, further studies at several bacteriocin concentrations are necessary in order to know the ability of GarA to form pores or inhibit septum formation through lipid II interactions.

L. garvieae is the main agent responsible for lactococcosis, an emergent disease causing hyperacute and hemorrhagic septicemia that affects different farmed and wild fish and crustacean species worldwide, from both fresh and marine waters, causing important economic losses (1). Usually, L. garvieae outbreaks in aquaculture are treated with antibiotics, but these are frequently ineffective, and their indiscriminate use is responsible for the appearance of antibiotic-resistant strains (1, 52). To overcome the problems resulting from the emergence of antibiotic-resistant L. garvieae strains (1, 52) and the limited number of antibiotics approved for use in aquaculture, several strategies, based mainly on the use of vaccines, probiotics, and bacteriocins, have been developed to fight lactococcosis (2–4).

Bacteriocins represent an alternative to existing antimicrobials due to their antibacterial activity against several relevant pathogenic bacteria in veterinary medicine and human health (53–55). Purified or partially purified bacteriocins can also be used for the treatment of target pathogenic bacteria, with a success rate even higher than that for the administration of bacteriocinogenic bacteria (56). The high specificity of GarA, which is active only against L. garvieae strains and lacks inhibitory activity against some beneficial bacteria, could have several important applications for both food and clinical uses. However, in order to confirm the narrow inhibitory spectrum of GarA, further studies using more strains or species will be necessary. Thus, the administration of the bacteriocin could prevent lactococcosis in fish by inhibiting the growth of pathogenic L. garvieae strains. In this work, we have shown that GarA is very active against the fish pathogen L. garvieae 8831, a strain isolated from diseased rainbow trout and responsible for most of the lactococcosis outbreaks in Spain (47). In addition, since GarA is not active against the lactic acid bacteria (LAB) tested, combined treatments with this bacteriocin and probiotic LAB could be devised. Probiotic treatments based on LAB have been proposed as a feasible alternative for fighting lactococcosis in aquaculture (57). To extend these results, the inhibitory activity of GarA against a wide range of recognized probiotics will be assayed in future experiments.

L. garvieae has also been associated with subclinical mastitis in cows and water buffalos (5, 6), causing important economic losses due to reduced milk production. In this work, we have shown that GarA is active against L. garvieae strains causing mastitis in cows and water buffalos and that it could therefore be used to prevent or treat subclinical mastitis due to this agent. In this context, both bacteriocin-producing lactococci and lactococcal bacteriocins, such as nisin or lacticin 3147, have been shown to possess a therapeutic effect on mastitis in lactating cows (58–62) and women (63).

In summary, this study describes the characterization of garvicin A, a novel linear non-pediocin-like one-peptide class IId bacteriocin with a narrow spectrum of inhibitory activity, limited to other strains of the same species. Its specific inhibitory spectrum, and its ability to inhibit septum formation, could be exploited for the prevention or treatment of infections caused by pathogenic strains of L. garvieae.