Raffinocyclicin is a novel plasmid-encoded circular bacteriocin produced by Lactococcus raffinolactis with broad-spectrum activity against many gram-positive food pathogens

ABSTRACT This study describes the discovery and characterization of raffinocyclicin, a novel plasmid-encoded circular bacteriocin, produced by the raw milk isolate Lactococcus raffinolactis APC 3967. This bacteriocin has a molecular mass of 6,092 Da and contains 61 amino acids with a three-amino acid leader peptide. It shows the highest identity to the circular bacteriocins bacicyclicin XIN-1 (42.62%), aureocyclicin 4185 (42.62%), and garvicin ML (41.53%). A broad inhibitory spectrum includes strains from Staphylococcus, Enterococcus, Streptococcus, Micrococcus, Lactobacillus, Leuconostoc, and Listeria, in addition to a pronounced inhibitory effect against Lactococcus and Clostridium. It displays low sensitivity to trypsin, most likely as a result of its circular nature. The raffinocyclicin gene cluster is composed of 10 genes: 6 core genes, genes encoding an accessory three-component ABC transporter (rafCDE), and a putative transcriptional regulator related to the MutR family. A lack of inhibitory activity in the cell-free supernatant combined with the pronounced activity of cell extracts suggests that the majority of raffinocyclicin is associated with the cell rather than being released to the extracellular environment. This is the first report of a bacteriocin produced by the L. raffinolactis species. IMPORTANCE The present study aimed to characterize raffinocyclicin, a novel circular bacteriocin produced by the lactic acid bacteria Lactococcus raffinolactis APC 3967. Bacteriocins are generally cationic and hydrophobic peptides with antimicrobial activity, which present diverse biotechnological properties of interest for the food industry. Raffinocyclicin inhibits a wide range of bacteria, including foodborne pathogens, and is stable against different treatments which suggest its potential as a natural biopreservative. Whole-genome sequencing and the genetic analysis of the raffinocyclicin gene cluster showed that it is encoded by plasmid that could be used in the future to transfer the ability to produce the bacteriocin to other lactic acid bacteria for industrial applications. These results together highlight the potential of this novel antimicrobial as a biopreservative to be used by the food industry.

of microorganisms used for millennia in the preservation of foods through fermen tation is the lactic acid bacteria (LAB).This bacterial group comprises several genera, including Lactococcus, lactobacilli, Leuconostoc, Pediococcus, Streptococcus, and Enterococcus.These bacteria can be found in various fermented foods, including yoghurt, sauerkraut, kefir, and cheese (3).LAB ferment sugars in the food matrix and produce lactic acid that can inhibit spoilage microorganisms, thus extending the shelf life of foods (1).However, this is not the only substance produced by these bacteria with a biopreservative property; many LAB also produce antimicrobial peptides known as bacteriocins (2).
Bacteriocins are defined as ribosomally synthesized multifunctional peptides produced by prokaryotes, which can have either a broad spectrum of antimicrobial activity (targeting a range of species) or a narrow spectrum (targeting closely related species and strains) (4,5).These peptides are generally cationic and hydrophobic, with the producing strain possessing immunity to its own bacteriocin.A number of classifi cation systems have been proposed (5)(6)(7)(8).However, they can be broadly divided into two main classes: class I peptides that undergo post-translational modification, such as circular bacteriocins, and class II unmodified peptides (9).Several studies have reported their potential as biopreservatives (10,11).Moreover, the class I bacteriocin nisin (E234) has been approved as a biopreservative since 1969.
Bacteriocins produced by LAB offer several biotechnological advantages over chemical preservatives.These include (i) their selfaffirmed GRAS (generally recognized as safe) status; (ii) they are generally non-toxic to eukaryotic cells; (iii) resistance to a range of pH, temperature, and food-associated enzymes; (iv) the ability to target pathogens and food spoilage bacteria; and (v) they can be encoded on mobile elements, such as conjugative plasmids or transposons, thus facilitating their mobilization to starter cultures (10,11).As for their applications in food, four main ways have been described: (i) direct application of the bacteriocin as a pure or partially purified substance; (ii) production in the food matrix by the bacteriocinogenic strain; (iii) incorporation of the antimicrobial in food packaging; and (iv) use of a fermented food ingredient derived from the bacteriocinogenic strain (12)(13)(14).
Circular bacteriocins are a group of bacteriocins that have been explored in recent years for their biotechnological potential.To date, this group of bacteriocins is composed of 23 peptides, with altitudin A being the most recently described in 2024 (15)(16)(17)(18)(19)(20)(21)(22)(23)(24).They can be divided into two groups: (i) peptides with a high isoelectric point and a high net charge (e.g., enterocin AS-48 and garvicin ML) or (ii) peptides with a considerably lower isoelectric point and a low net charge (e.g., butyrivibriocin AR10 and paracyclicin) (18).One main characteristic that makes circular bacteriocins so promising for food preserva tion and safety is that they are highly resistant to pH, temperature, and proteolytic action.This characteristic is due to their highly stable circular and globular conformation (25).However, the application of this group of bacteriocins is limited due to the lack of knowledge about their biosynthetic pathways and biopreservative and safety potential compared to other classes.
This study describes the isolation and characterization of raffinocyclicin, a circular bacteriocin produced by the LAB strain Lactococcus raffinolactis APC 3967.

Antimicrobial spectrum of Lactococcus raffinolactis APC 3967
The antimicrobial activity of L. raffinolactis APC 3967, a strain isolated from bulk tank milk, was evaluated by the spot-on-lawn assay and showed a broad spectrum of activity, with 22 of 31 indicator strains inhibited by the producer (Table 1).Sensitive strains included members of the following genera: Staphylococcus, Enterococcus, Streptococcus, Micrococ cus, Lactococcus, Lactobacillus, Leuconostoc, Listeria, and Clostridium.No inhibition was observed against the gram-negative Escherichia sp.UCC.The two most sensitive species identified in this screening were Clostridium perfringens and Lactococcus lactis.

Sensitivity of raffinocyclicin to proteases and NaOH
The antimicrobial activity of the strain L. raffinolactis APC 3967 was resistant to NaOH 0.2 M, partially sensitive to trypsin (retaining 73% of the initial activity after treatment), and highly sensitive to proteinase K (retaining only 7% of the initial activity after treatment), confirming a proteinaceous nature (Table 2).

Colony mass spectrometry
The observed molecular mass of 6,092 Da did not match the mass of any known bacteriocin, suggesting that the antimicrobial activity was due to a potentially novel peptide (Fig. 1).

Genomic analysis
To identify the gene(s) involved in the production of the antimicrobial molecule, the whole genome of L. raffinolactis APC 3967 was sequenced.The assembly of long and short reads resulted in a complete genome with a size of 2,348,986 bp (sequence coverage: short reads 50×, long reads 35×) with a GC content of 39.7%.The genome consists of a chromosome (2,258,934 bp) and three plasmids (50,437, 33,572, and 6,043 bp; Fig. 2).These extrachromosomal DNA elements harbor genes associated with plasmid functions, including a RepB family replication protein and/or other proteins, confirming their plasmid-like nature.
After annotation using Prokaryotic Genome Annotation Pipeline (PGAP), the genome was analyzed using BAGEL4 and Antismash 7.0 for the presence of bacteriocin gene clusters.Two gene clusters were found: (i) one related to a putative class II lactococcin 972-like bacteriocin, found with both programs and (ii) a circular garvicin ML-related bacteriocin, found only with BAGEL4.The lactococcin 972-like bacteriocin is encoded on the chromosome, and the putative circular bacteriocin is encoded on the ~50-kb plasmid pRaff01 (Fig. 2).
To determine if the strain produces either or both putative bacteriocins, the theoreti cal masses of their core peptides were compared with the 6,092 Da mass obtained from colony mass spectrometry.Based on the cleavage site of the closely related lactococcin 972, the lactococcin 972-like bacteriocin was predicted to be 7550.02Da (26) (Fig. 3).Using the cleavage site of garvicin ML, the core peptide of the putative circular  bacteriocin was calculated as 6110.17Da (27) (Fig. 4A).However, circular bacteriocins undergo a loss of 18 Da after the head-to-tail cyclization, between leucine 1 and tryptophan 61 in this instance, culminating in a predicted final mass of 6092.17Da.This precisely matches the mass detected by the colony mass spectrometry, confirming that the strain is producing a novel circular bacteriocin herein named raffinocyclicin.There fore, subsequent analysis focused on this bacteriocin.The nucleotide sequence of the surrounding sequence reveals the presence of the canonical ribosome-binding site AGGAGG placed appropriately upstream of the structural gene for raffinocyclicin in the operon (Fig. 4B).A graphical representation of the core peptide is presented in Fig. 4C.In silico analysis of pRaff01 revealed that the novel circular bacteriocin gene cluster is ~7.5 kb in length and is composed of 10 genes (rafX, rafI, rafT2, rafT1, rafA, rafB, rafR, rafC, rafD, rafE) across both DNA strands (Fig. 2D and 5).The principal physiochemical characteristics, subcellular localization, and highest similarity hits of each gene product are described in Table 3.When compared to other circular bacteriocins, the raffinocy clicin gene cluster contains more genes than previously described clusters for similar bacteriocins.This is due to the presence of a second putative membrane protein and an accessory multi-component ABC transporter.The same genes can be found in the gene clusters of aureocyclicin 4185, carnocyclin A, enterocin AS-48, and garvicin ML.With the exception of garvicin ML, which has 9 genes, the other 3 circular bacteriocins have gene clusters comprising 10 genes.
The gene rafA encodes the core peptide of the bacteriocin of this genetic cluster, which is 64 amino acids in length and includes a 3-amino acid leader sequence and a mature peptide of 61 amino acids.This leader sequence (MFD) is the same as that of garvicin ML, which is a closely related circular bacteriocin.The gene rafB encodes a membrane protein that contains the DUF95 domain of unknown function.This type of protein has been found in all circular bacteriocin gene clusters described in the literature (15)(16)(17)(18)(19)(20)(21)(22)(23)(24).The gene rafR, on the opposite DNA strand, encodes a positive transcriptional regulator belonging to the MutR family, which could potentially be involved in regulat ing raffinocyclicin biosynthesis.The genes rafC, rafD, and rafE are thought to encode a three-component ABC transporter often found in the genetic clusters of the circular bacteriocins (aureocyclicin 4185, carnocyclin, circularin A, enterocin AS-48, and garvicin ML) with a putative role in transport and immunity (18).Four genes were found upstream of the core peptide: rafT1, rafT2, rafI, and rafX.The two genes rafT1 and rafT2 encode two putative membrane proteins with low similarity to other membrane proteins associated with circular bacteriocins.The gene rafI encodes a putative dedicated immunity protein with a short, hydrophobic, and cationic product of 52 amino acids.These biochemical characteristics are shared between the immunity protein of all circular bacteriocins (18).Lastly, the gene rafX encodes a cytoplasmatic ATP-binding cassette domain-containing protein.

Mature peptide alignment
The mature peptide amino acid sequences of all 23 circular bacteriocins identified to date were aligned with the mature sequence of raffinocyclicin (Fig. 6A).To improve the alignment quality, the first three amino acids of the group II bacteriocins were permuted to the end of the sequence as previously described (20).The change of the amino acid position does not change the relevance of the alignment due to the circular nature of these peptides.The homology between peptides is very low.Assuming that the numbering of amino acids remains the same after circularization, except for group II, the first amino acid of all circular peptides is hydrophobic in nature (valine, methionine, leucine, or isoleucine).Another observed characteristic is that the last amino acid of most peptides is aromatic (phenylalanine, tyrosine, and tryptophan), with the only exceptions being carnocyclin A, garvicin ML, and pallidocyclin that have leucine, alanine, and alanine, respectively.Lastly, most of the positively charged amino acids (lysine, arginine, and histidine) of all circular bacteriocins seem to cluster together in just one region of the peptide.
The phylogenetic tree of the mature core peptides reveals that the closest relatives of raffinocyclicin are bacicyclicin XIN-1 and aureocyclicin 4185 (both with 42.62% identity), followed by garvicin ML (41.53% identity; Fig. 6B).It is essential to highlight that group I and group II form two distinct branches in the phylogenetic tree.

Bacteriocin purification
In order to confirm that the molecular mass found by colony mass spectrometry was responsible for the antimicrobial activity observed in the spot-on-lawn assay, the peptide was purified by a two-step protocol.It is important to highlight that the cell extract was used since no inhibitory activity was observed in the cell-free supernatant (CFSN).Following extraction from the cell pellet using IPA, the peptide was semipurified by solid-phase extraction (SPE) and then fractionated on a semi-preparative Jupiter C4 RP-HPLC column.The presence of the antimicrobial activity was monitored by testing the fractions obtained against L. lactis HP by well diffusion assay.The results reveal that fraction 74 obtained from HPLC presents the highest activity against the indicator strain (Fig. 7A).This finding correlates with the peak (~ 15 mV)-outlined in red on the chromatogram.MALDI-TOF (matrix-assisted laser desorption/ionization coupled to timeofflight) analysis of the active fraction reveals the presence of a peptide with the expected molecular mass for raffinocyclicin.Both doubly charged [M + 2H] 2+ and singly charged [M + H] + species of the peptide are present with masses of 3046.72 Da (±1 Da) and 6093.08Da (±1 Da), respectively (Fig. 7B).

Pangenomic analysis
Pangenome analysis for the strain was performed to investigate the phylogenetic relationship of the genome of L. raffinolactis APC 3967 with other publicly available L. raffinolactis genomes described in the literature.The strain L. laudensis DSM 28961, a representative of a closely related species, was used as an outlier for this in silico analysis.First, all genomes were annotated with Prokka, with the resulting output subjected to pangenome analyses using Roary using a minimum percentage of amino acid identity for a positive hit at 95%.Those genes present in all genomes were considered core genes.
A phylogenetic tree of the L. raffinolactis strains was created from the core gene alignment output obtained with Roary.The results reveal that the strain L. raffinolactis APC 3967 is more related to strains Lr_19_5, ERR5094873, 3042, Lr_19_14, Lr_19_4S, WiKim0068, and Lr_19_7, compared to other genomes examined (Fig. 8A).From these results, all L. raffinolactis genomes were investigated for the presence of the raffinocycli cin gene cluster to determine if there is a correlation between the strain location on the phylogenetic tree and bacteriocin production.Four of the 15 genomes (Lr_18_12S, 3039, ATCC 43920, and Lr_19_14S) encode the circular bacteriocin gene cluster.However, no correlation was found between the presence of the raffinocyclicin gene cluster and strain location in the phylogenetic tree (Fig. 8A).
The four strains encode an identical raffinocyclicin core peptide, but differences in the gene cluster can be observed in some strains (Fig. 8B).The strain L. raffinolactis ATCC 43920 seems to possess a shorter gene cluster with a truncated version of the gene rafT2 and an absence of the genes rafI and rafX.The strains L. raffinolactis 3039 and Lr_18_12S have an additional open reading frame (ORF) in the gene cluster with no NCBI database hits.No other modifications were observed in the other strains.

DISCUSSION
The circular bacteriocins are a group of peptides that have been gaining increased attention in the last few years, with the discovery of new representatives and increased interest in their potential biotechnological applications.This class of peptides has an unusual post-translational modification, namely head-to-tail cyclization, which leads to higher stability of the peptide (against pH, temperature, and proteases, among other conditions) compared to linear peptides (28).These properties make them desirable candidates for drug design and as biopreservatives.However, this group of peptides is still underexplored compared to other bacteriocin classes, with only 23 such peptides reported in the literature to date.This work presents a novel circular bacteriocin named raffinocyclicin, the first reported bacteriocin for the L. raffinolactis species.
Regarding the antimicrobial activity, the strain L. raffinolactis APC 3967 has a broad spectrum of activity, inhibiting most of the indicators used for the spot-on-lawn assay (22/31).The highest inhibitory activity was found against L. lactis and C. perfringens.It is important to highlight that L. lactis is a good indicator in terms of bioprospecting for circular bacteriocin producers due to its sensitivity to diverse circular bacteriocins, such as carnocyclin A (29); pumilarin (30), plantaricyclin A (31), uberolysin (32), garvicin ML (27), plantacyclin B12AG (20), leucocyclicin Q, and lactocyclicin Q (33).Indeed, the narrow-spectrum plantaricyclin A inhibited all five strains of L. lactis tested, emphasizing the utility of this species as an indicator in future screens for circular bacteriocins (31).In this study, L. raffinolactis APC 3967 could also inhibit different pathogen species, such as C. perfringens, L. monocytogenes, E. faecium, E. faecalis, and S. agalactiae.The inhibitory spectrum of this bacteriocin suggests its potential for application as a food biopreservative or as an antimicrobial agent in the clinic.
The proteinaceous nature of the antimicrobial activity of L. raffinolactis APC 3967 was confirmed following the loss of over 90% of its activity when treated with protei nase K. Furthermore, the inhibitory activity remained unchanged after NaOH treatment,  presented a low-sensitivity profile (30); and carnocyclicin A had its antimicrobial activity completely abolished by protease treatment (29).This proteolytic resistance of some circular bacteriocins against proteases is not due to the lack of cleavage sites but probably due to the globular three-dimensional conformation that makes such sites inaccessible for the enzyme (27,31).Analysis of the raffinocyclicin gene cluster revealed 10 ORFs, 5 genes downstream of the core peptide and 4 genes upstream of it.All predicted genes necessary for bacteriocin biosynthesis are present in the cluster, such as the precursor peptide (rafA), a dedicated immunity protein (rafI), a membrane protein containing a DUF95 domain (rafB), an ABC transporter (rafX), and membrane proteins (raf T1 and rafT2) (18).However, a putative three-component ABC transporter (rafCDE) is also encoded and is most probably associated with an accessory function as a secondary transporter.This hypothesis is supported by studies with garvicin ML, where the deletion of the corresponding genes in the operon (garFGH) did not affect bacteriocin production or immunity (34).For the corresponding genes of enterocin AS-48 (as-48EFGH) and carnocyclin A (cclEFGH), deletion leads to a reduction of bacteriocin immunity and/or production (35,36).The most unusual gene found in the raffinocyclicin gene cluster is the gene rafR.Another example in this class of peptide of a putative transcriptional regulator was the protein LcyR encoded by a gene present in the gene cluster of leucocyclicin Q (37).The putative product of this gene is a positive transcriptional regulator of the MutR family, which is found in some bacteriocins produced by Strep tococcus mutans to ensure the transcription of the core peptide and other genes of the cluster (38).These data suggest that the gene rafR could play a significant role in raffinocyclicin biosynthesis.
The alignment of the mature sequence of all circular bacteriocins (Fig. 6A) revealed a pattern in that the majority of the positively charged amino acids (lysine, arginine, and histidine) are grouped in one region of the peptide.This disproportional positive charge distribution through the peptide was also observed for enterocin AS-48.Previous studies proposed that the positive charge cluster plays a major role in the binding of the peptide to the membrane and its inhibitory activity (28).Similar asymmetrical distribution can be found in all circular bacteriocins, which leads us to propose that this region probably first interacts with the cellular surface of the target microorganism.This information could help better understand the mechanism of action of circular bacteriocins, and it should be further investigated.
A two-step method was applied to purify the peptide.Due to the lack of activity in the cell-free supernatant, the cell pellet was used as the primary source for purification.The masses found in the colony mass spectrum and purified peptide spectrum are both 6,092 ±1 Da, which confirms the hypothesis that raffinocyclicin has a three amino acid leader sequence (MFD) and that it undergoes head-to-tail circularization leading to a loss of 18 Da (6,110 − 18 = 6,092 Da).Future analysis must be done in order to support the hypothesis of the circularization event proposed from the purified bacteriocin mass spectrum.
The lack of activity in the CFSN and the activity associated with the cell extract (Fig. 1) suggest that the majority of the bacteriocin is associated with the cell.A similar phenotype has been observed previously for the lantibiotic bovicin HC5 (39).This characteristic could lead to a more stable antimicrobial activity of the bacteriocin in a complex ecosystem, such as the gut, with cell-associated peptides displaying greater resistance to proteases and peptidases produced by competing microorganisms (39).However, it is important to highlight that the circular bacteriocins present high resistance to those enzymes, as already stated.However, this hypothesis needs further evaluation for this group of peptides.
Another example of this behavior was observed with listeriolysin S, where the bacteriocin remains associated with the cell and only displayed its inhibitory activity in a contact-dependent manner.Unlike raffinocyclicin and bovicin HC5, listeriolysin S does not show inhibitory activity when not associated with the membrane of metabolically active producing bacterial cells (40).Altogether, those results propose the concept that some prokaryotes could use bacteriocins for close-contact combat in a competitive environment.This hypothesis needs to be further investigated for circular bacteriocins.
No correlation was found between the alignment of core genes and bacteriocin presence on the L. raffinolactis genomes, which leads to the conclusion that the gene cluster could be encoded by a mobile genetic element, such as a plasmid, which is the case of the strain APC 3967.The results show two positive hits with plasmids of the strains Lr_19_4S (query cover 36%; identity 99.96%; CP050535.1)and Lr_18_12S (query cover 22%; identity 97.80%; CP047632.1).Both strains, Lr_19_4S and Lr_18_12S, also present the complete raffinocyclicin gene cluster (Fig. 8B).The presence of the gene clusters of circular bacteriocins on plasmids has already been reported in the literature, such as enterocin AS-48, acidocin B, aureocyclicin 4185, paracyclicin, gassericin A, plantaricyclin A, and plantacyclin B12AG (41).

Conclusion
Raffinocyclicin is a novel circular bacteriocin produced by the strain L. raffinolactis APC 3967 with a broad inhibitory spectrum, which undergoes head-to-tail cyclization between Leu1 and Trp61 and has a molecular mass of 6,092 Da.The plasmid-encoded gene cluster of this peptide includes 10 ORFs, and one of them, an unusual gene, rafR, encodes a putative-positive transcriptional regulator.The production of raffinocyclicin by a LAB opens possibilities for biotechnological applications in the food industry due to its GRAS status, either as a starter or safety culture in fermentation processes or as a purified biopreservative.More studies are necessary to evaluate this feature entirely.This is the first report of a bacteriocin produced by the L. raffinolactis species.

Antimicrobial activity
The antimicrobial activity of the strain L. raffinolactis APC 3967 was evaluated on M17 medium by the agar-spot test as described by Giambiagi-deMarval et al. (42) with minor modifications (42).Briefly, 5 µL of an overnight growth suspension of the producer strain was spotted on the surface of the agar plate and incubated overnight at 30°C.Cells were inactivated by chloroform and overlaid with 3 mL soft agar (0.75% wt/vol) containing the target strain (∼7.0 log CFU/mL), and these plates were incubated overnight.The growth conditions of every target strain used in this assay are outlined in Table 1.The interpretation of inhibition was performed by measuring the true halo of the inhibitory zone (total halo -spot size = true halo) and was classified into one of four groups: (i) no inhibition (0 mm); (ii) weak inhibition (0.5-5 mm); (iii) moderate inhibition (>5-≤10 mm); and (iv) strong inhibition (>10 mm).These assays were performed in triplicate.

Evaluation of the effects of proteases and NaOH on the antimicrobial activity
The effects of proteinase K (1 mg/mL; Sigma-Aldrich, St Louis, USA), trypsin (1 mg/mL; Merck KgaA, Darmstad, DE), and 0.2 M NaOH on antimicrobial activity were evaluated on agar plates by the methods described previously (42).Briefly, 5 µL of an overnight growth suspension of the strain L. raffinolactis APC 3967 was spotted four times on a plate and incubated at optimal conditions.Afterward, the cells were inactivated by chloroform, and 40 µL of the corresponding treatment (protease or NaOH 0.2 M) was spotted around the producer strain.The control was treated with 10 mM PBS, pH 7.4.The plate was incubated at 37°C for 4 h to favor protease activity.The plate was then overlaid with L. lactis HP and incubated at 30°C for 24 h.Results were interpreted by measuring the true halo of the sample, and the following equation calculated the final values of the residual activity: (true halo of treated condition/true halo of the control) × 100 = residual activity (%).

Colony MALDI-TOF mass spectrometry
To determine the molecular mass of the bacteriocin produced by the strain, colonies isolated from the plate were mixed with 50 µL 70% propan-2-ol (IPA) 0.1% TFA, vortexed three times, and centrifuged at 15,000 rpm (21,000 rcf ) for 20 seconds.MALDI-TOF mass spectrometry was performed on the cell-free extract using an Ultraflex MALDI-TOF mass spectrometer (Bruker, Bremen, Germany).Specifically, 0.5 µL aliquot of matrix solution [α-cyano 4-hydroxy cinnamic acid, 10 mg/mL in acetonitrile-0.1% (vol/vol) trifluoroacetic acid] was deposited onto the target and left for 20 seconds before being removed.The residual solution was air dried, and 0.5 µL sample solution was deposited onto the pre-coated sample spot.Furthermore, 0.5 µL of matrix solution was added to the deposited sample and allowed to air-dry.The sample was subsequently analyzed in positive-ion reflectron mode, and peptide masses were compared to a bacteriocin database to identify putative bacteriocins (43).

Circular bacteriocin core peptide alignment
The core peptide sequences of all 23 circular bacteriocins and raffinocyclicin were aligned using MAFFT v7.4.89 (54).The permutation of the three amino acids, from the beginning of the sequence to the end of the sequence, for the peptides that belong to group II (acidocin B, butyrivibriocin AR10, gassericin A, paracyclicin, plantacyclin B21AG, and plantaricyclin A) was done to improve the alignment between the sequences (20).MEGA X was used to construct the phylogram using the maximum likelihood method using the Jones-Thornton-Taylor model (55,56).The resulting phylogram was visualized using the Interactive Tree of Life (iTOL) v6.7.4 (https://itol.embl.de/)(57).

Bacteriocin purification
A 900 mL culture of L. raffinolactis APC 3967 was grown overnight at 30°C in M17 broth.The culture was centrifuged at 8,280 g for 20 minutes at 10°C, and cells were separated from the supernatant.The cell pellet was resuspended in 200 mL of 75% propan-2-ol and stirred at room temperature for 3-4 hours to extract the bacteriocin from the cell mass.The suspension was centrifuged at 8,280 g for 20 minutes, and the supernatant was retained.The IPA from the supernatant was evaporated using a rotary evaporator (Buchi Labortechnik AG, Flawil, Switzerland), and the sample was applied to a 5-g (20 mL) Strata C18-E SPE column (Phenomenex, Cheshire, UK) pre-equilibrated with methanol and water.The column was washed with 40 mL of 40% ethanol, and the inhibitory activity was eluted in 30 mL IPA.The organic solvent was removed from the sample via rotatory evaporation.The concentrated sample was applied to a semi-preparative Jupiter C4 (10 × 250 mm, 300 Å, 5 µm) RP-HPLC column (Phenomenex, Cheshire, UK) running an acetonitrile and propan-2-ol gradient described as follows: 5%-55% buffer B and 0%-5% buffer C over 25 minutes followed by 55%-19% buffer B and 5%-65% buffer C over 60 minutes, 19%-5% buffer B and 65%-95% buffer C over 5 minutes where buffer B is 90% acetonitrile 0.1% TFA and buffer C is 90% propan-2-ol 0.1% TFA.Eluent was monitored at 214 nm, and fractions were collected at 1-minute intervals.Fractions were assayed on L. lactis HP indicator plates, and active fractions were assayed for the antimicrobial mass of interest using MALDI TOF mass spectrometry.

Statistical analysis
When relevant, the data were expressed as the mean value ± standard deviation.The data were analyzed by Microsoft Excel.

FIG 1
FIG 1 Colony matrix-assisted laser desorption/ionization coupled to timeofflight mass spectrum revealing the molecular mass of raffinocyclicin produced by L. raffinolactis APC 3967 (6,092 Da).

FIG 4 (
FIG 4 (A) Alignment of the precursor peptides of raffinocyclicin and garvicin ML, the leader sequences are underlined.Gap (−), identical amino acids (*), conservative substitutions (:), and weak conservative substitutions (.).The leader sequences are underlined.(B) The nucleotide sequence of the raffinocyclicin propeptide.The cleavage site is highlighted by the dashed line between residues 3 and 4. RBS, putative ribosome-binding site-7 bp upstream of the start codon.*, stop codon.(C) Predicted circular structure of raffinocyclicin.The yellow amino acids correspond to the leader sequence of the bacteriocin.

FIG 5
FIG5 The proposed gene cluster of raffinocyclicin.

FIG 6 (
FIG 6 (A) Amino acid alignment of the mature circular bacteriocins (MAFFT v7.4.89).Gap (−), identical amino acids (*), and conservative substitutions (:).Dashed red lines are used to show the region of the peptide where most of the positively charged amino acids are found (H, K, and R).(B) Maximum likelihood phylogenetic tree of the mature circular bacteriocins using MAFFT for the alignment.Bootstrapped *1,000 replicates.The green boxes were used to highlight raffinocyclicin, garvicin ML, bacicyclicin XIN-1, and aureocyclicin 4185.*, net charge predicted for the bacteriocin linear form using GeneScript Peptide Property Tool.

FIG 7 (
FIG 7 (A) HPLC analysis of the cell extract of L. raffinolactis APC 3967 with fraction 74, highlighted by the red circle, and showing antimicrobial activity against L. lactis HP. (B) Detection of the theoretical mass of raffinocyclicin by MALDI-TOF mass spectrum from fraction 74.Expected mass: 6,092.17Da (±1 Da).

FIG 8 (
FIG 8 (A) Approximate maximum likelihood phylogenetic tree of non-redundant genomes belonging to L. raffinolactis.The tree showing all 17 genomes (16 L. raffinolactis and 1 L. laudensis) was computed using a core gene alignment file generated using Roary, bootstrapped *1,000 replicates.The strain L. laudensis DSM 28961 was used as an outlier.(B) Raffinocyclicin gene clusters encoded by different L. raffinolactis strains.

TABLE 1
Growth conditions of the indicator strains and inhibitory spectrum of raffinocyclicin in the spot-on-lawn assay a,b

TABLE 2
Residual antimicrobial activity after the different treatments a

TABLE 3
Bioinformatics analysis of the raffinocyclicin gene cluster (27)onent ruling out that the activity was a consequence of organic acids.Exposure to trypsin revealed that the antimicrobial activity has low sensitivity to this enzyme, given that 73% of residual activity remained following treatment.Interestingly, leucocyclicin Q, lactocyclicin Q (33), and garvicin ML(27)were resistant to proteolytic activity; pumilarin