Crystal structure and site-directed mutagenesis of circular bacteriocin plantacyclin B21AG reveals cationic and aromatic residues important for antimicrobial activity

Plantacyclin B21AG is a circular bacteriocin produced by Lactiplantibacillus plantarum B21 which displays antimicrobial activity against various Gram-positive bacteria including foodborne pathogens, Listeria monocytogenes and Clostridium perfringens. It is a 58-amino acid cyclised antimicrobial peptide, with the N and C termini covalently linked together. The circular peptide backbone contributes to remarkable stability, conferring partial proteolytic resistance and structural integrity under a wide temperature and pH range. Here, we report the first crystal structure of a circular bacteriocin from a food grade Lactobacillus. The protein was crystallised using the hanging drop vapour diffusion method and the structure solved to a resolution of 1.8 Å. Sequence alignment against 18 previously characterised circular bacteriocins revealed the presence of conserved charged and aromatic residues. Alanine substitution mutagenesis validated the importance of these residues. Minimum inhibitory concentration analysis of these Ala mutants showed that Phe8Ala and Trp45Ala mutants displayed a 48- and 32-fold reduction in activity, compared to wild type. The Lys19Ala mutant displayed the weakest activity, with a 128-fold reduction. These experiments demonstrate the relative importance of aromatic and cationic residues for the antimicrobial activity of plantacyclin B21AG and by extension, other circular bacteriocins sharing these evolutionarily conserved residues.

Bacteriocins are a group of ribosomally-synthesised antimicrobial peptides produced by both Gram-negative and Gram-positive bacteria. They generally confer antimicrobial activity against bacterial species and/or strains closely related to the bacteriocin producers. Many studies [1][2][3][4] have suggested that these antimicrobial peptides have potential as natural food preservative. Bacteriocins have also been identified as possible next generation antibiotics to combat multiple-drug resistant pathogens 3,5 . In recent years, bacteriocins from Gram-positive bacteria, especially lactic acid bacteria (LAB) have attracted interest because they are generally regarded as safe (GRAS) for human consumption and are thought to have a broader antimicrobial spectrum than bacteriocins produced by Gram-negative bacteria 6 . Furthermore, Gram-positive bacteriocins are active against foodborne pathogens, making them important candidates for controlling food spoilage and pathogenic bacteria in the food and pharmaceutical industries 6 .
Bacteriocins produced by Gram-positive bacteria are divided into three main classes: class I-modified, class II-unmodified and class III-large, heat labile 7 . Of these, circular bacteriocins (class I) have gained attention due to their unique characteristics of high thermal and pH stability, as well as resistance to degradation by many proteolytic enzymes. The covalently linked N-and C-termini form a structurally distinct cyclic peptide Scientific Reports | (2020) 10:17398 | https://doi.org/10.1038/s41598-020-74332-1 www.nature.com/scientificreports/ The protein has an overall net charge of + 3 at physiological pH, with four cationic residues located in helix α1 (Lys 15 and Lys 19 ), helix α4 (Arg 6 ) and the loop connecting α1/α4 (His 11 ), and one anionic residue located in helix α1 (Asp 22 ) (Fig. 1c, upper panel). These charged residues are all displayed on the surface of the plantacyclin B21AG structure. The electrostatic potential surface map of plantacyclin B21AG reveals two cationic patches separated by an anionic strip (Fig. 1c, lower panel).

Structural comparisons of circular bacteriocins.
To better understand the similarities and differences between known structures of circular bacteriocins, structural comparisons were performed between plantacyclin B21AG and other bacteriocin 3D structures elucidated to date, namely enterocin AS-48, carnocyclin A and enterocin NKR-5-3B (family I) and acidocin B (family II). We note that the acidocin B structure was determined using the bacteriocin embedded in SDS micelles 28 , whereas the other bacteriocin structures were determined under aqueous conditions 13,34,44 . These circular bacteriocins adopt a common 3D structure comprising four or five helices folded into a globular bundle enclosing a hydrophobic core 11 (Fig. 2, left panel) 13,28,34,44 . The orientation of the helices is similar within the members of each family, with the exception of enterocin AS-48 which has five rather than four α-helices. Notably, the circularisation point for all these bacteriocins is located within a helical structure which contains mostly hydrophobic residues 36 . Analysis of solvent exposed residues using GETAREA 45 reveals that the N-to C-linkage of all these bacteriocins except the micelle embedded acidocin B is buried in the protein core (the ratio of side chain surface area to the average solvent-accessible surface area of the first and last amino acid, respectively is less than 20%). Unlike saposins and saposin-like peptides which are stabilised by disulfide bonds between cysteine residues 46 , the bacteriocin helical fold appears to be stabilised by hydrophobic side chain interactions upon peptide circularisation 44 . Martin-Visscher et al. 44 proposed that hydrophobic residues close to the N-C linkage may play a role in the interaction between the linear peptide and the cyclisation enzyme, helping to bring the termini into close proximity for cyclisation to take place. A combination of circular backbone and hydrophobic core is thought to contribute to the stability of circular bacteriocins 13 . Surface characteristics of circular bacteriocins. The molecular surface of the circular bacteriocins is amphipathic: one face of the molecule has cationic and anionic patches, and the other surface is uncharged Table 1. Data collection and refinement statistics for the crystal structure of plantacyclin B21AG. The number of crystals used for structure determination and refinement is one. a Highest resolution shell is shown in parenthesis. www.nature.com/scientificreports/ or hydrophobic (Fig. 2, right panel). Generally, family I circular bacteriocins are more cationic than family II circular bacteriocins (Table 2). Noteworthy is the cationic region located close to the N-and C-ligation point (α1 and α4). This cationic region is present in enterocin AS-48, carnocyclin A and enterocin NKR-5-3B. Eight lysine residues are located along the stretch of α4 and α5 in enterocin AS-48. Similarly, four lysine residues are located along α3 and α4 of carnocyclin A and enterocin NKR-5-3B (Fig. 2, right panel). The surface representation of the circular bacteriocins presented in Fig. 2 suggests that the charge distribution is conserved among circular bacteriocins 13 . The cationic surface patches are thought to be involved in an initial electrostatic interaction between the peptide and the negatively charged phospholipid bilayer of target cell membranes 8,13,36 .

Data collection
Binding of the cationic regions onto the target membrane may result in destabilisation of the membrane, potentially enabling peptide insertion 44 . However, it has also been demonstrated that electrostatic interaction alone is insufficient for antimicrobial activity. A 21-residue peptide fragment of enterocin AS-48 containing the cationic putative membrane interacting region exhibited competitive membrane binding 47 but did not show antibacterial activity, suggesting that other physicochemical properties of the bacteriocins may be required for antimicrobial action.
Most of the bacteriocin anionic residues are located in close proximity to the cationic patches ( Fig. 2, right panel). Whilst the role of anionic residues are generally overlooked in the context of circular bacteriocins, studies have shown that mutation of anionic residues in other classes of bacteriocins and/or antimicrobial peptides resulted in reduced bactericidal potency and altered target cell specificity 48,49 . Replacing aspartic acid at position 17 with the glutamic acid strongly enhanced the antimicrobial activity of the pediocin-like bacteriocin sakacin P. This result suggests that the anionic residues interact in a structurally specific and restricted manner with a cationic region on the target cell or on the peptide itself 50 . Given that circular bacteriocins have been found to kill through disruption of proton motive force and pore formation [51][52][53] , it is likely that their conserved structural similarities will result in the placement of negative charges near the pore so as to facilitate pore selectivity and cation flow out of the target cell by reducing the electrostatic energy profile and increasing selectivity for divalent cations such as Ca 2+54 . These findings suggest that anionic residues are as important as cationic residues in terms of target specificity of the bacteriocins.
The Grand Average of Hydropathy (GRAVY) Index 55 calculation reveals that family II circular bacteriocins are more hydrophobic than those in family I-except Carnocyclin A ( Table 2). The hydrophobicity of plantacyclin B21AG is evident in that it selectively dissolves into butanol fractions during purification. Some hydrophobic residues are exposed at the surface of the molecules (Fig. 2, right panel). The hydrophobicity of these peptides is thought to be crucial for creating pores in the bacterial membrane, especially for family II bacteriocins as they are less cationic 28,46 (Table 2). Permeation of the cell membrane by these peptides causes leakage of ions, dissipation of membrane potential and eventually cell death 8 . The molecular mode of action of enterocin AS-48 has been widely studied and is the best understood of the circular bacteriocins. A model has been proposed 34 such that the peptide in the form of a water-soluble dimer approaches the membrane surface of target bacteria through electrostatic interaction. Upon membrane interaction, each protomer within the water-soluble dimer rotates 90° and rearranges such that the hydrophobic helices become solvent accessible. The transition of AS-48 from water-soluble dimer to membrane-bound dimer allows the bacteriocin to insert into the bacterial membrane. This insertion alters the membrane potential, causing pore formation and cell leakage 34,47,52 . Collectively, these studies 34,47,52 have shown that cationic patches and hydrophobic patches play an important role in the mechanism of action of bacteriocins.
Structural alignment of circular bacteriocins. Structural alignment using the align function in PyMOL 56 revealed that the three-dimensional structure of family II plantacyclin B21AG aligned well with family I enterocin NKR5-3B, with a r.m.s.d of 2.47 Å across 30 C α atoms. For both structures, the helices α1, α2 and α4 are similar in length and align in parallel in a similar orientation. α3 of enterocin NKR5-3B is twice the length of α3 of plantacyclin B21AG but is oriented similarly (Fig. 3). The alignment of plantacyclin B21AG with enterocin NKR5-3B suggests that family I and family II bacteriocins could share a conserved three-dimensional structure with a very similar core fold, despite low sequence identity (15.6%) (Fig. 4). In contrast, there is poor structural alignment between plantacyclin B21AG and enterocin AS-48 with r.m.s.d of 4.54 Å across 45 C α atoms ( Fig. 3) (sequence identity 11.4%, Fig. 4). AS-48 also has a longer sequence than plantacyclin B21AG (70 residues compared with 58) and forms five α-helices rather than four (Fig. 2). Similarly, plantacyclin B21AG and carnocyclin A do not align well, with r.m.s.d of 4.33 Å across 45 C α atoms ( Fig. 3) (sequence identity 16.7%, Fig. 4). Hydrophobicity of plantacyclin B21AG. In this figure, the side chain atoms of hydrophobic residues (Ala, Ile, Leu, Phe, Pro, Gly, Trp and Val) are coloured in yellow. All other atoms of the same residues and all atoms of non-hydrophobic residues are shown in grey. Upper panel: Ribbon diagram illustrating the compact, hydrophobic core of plantacyclin B21AG. Hydrophobic side chains are drawn as sticks; Lower panel: Surface representation showing the solvent exposed hydrophobic side chains. (c) Ribbon diagram (upper panel) and electrostatic potential (lower panel) depicting amphipathicity of plantacyclin B21AG at physiological pH. In the ribbon diagram, side chain atoms of the cationic and anionic residues are depicted in blue (Arg, Lys and His) and red (Asp), respectively. All other atoms of the same residues and all atoms of neutral residues are shown in grey. Electrostatic potential calculated by APBS function 84 using PDB2PQR version 2.1.1 85 and surface map generated with Chimera 1.14 86 . Cationic, anionic and neutral residues are depicted in blue, red and white, respectively. Key residues are labelled with single amino acid codes and residue numbers. The helical orientation of the four/five α-helices is shown as α1 (blue), α2 (green), α3 (yellow), α4 (red) and α5 (orange). The black arrows indicate the orientation of the α helices. The dotted black lines represent the linkage between the Nand C-terminal. Right panel: Surface representation of the protein structures depicting side chain atoms of hydrophobic residues (yellow), cationic residues (blue), anionic residues (red) and polar residues (grey) at physiological pH. Lysine residues are indicated with single amino acid codes and residue numbers.  www.nature.com/scientificreports/ Plantacyclin B21AG shares 65% sequence identity with acidocin B (Fig. 4), and both belong to the circular bacteriocin family II. As indicated above, the NMR structure of acidocin B was determined in the presence of SDS micelles 28 . Not surprisingly, the structure of plantacyclin B21AG (determined in aqueous conditions) did not align well with that of acidocin B, giving a r.m.s.d. of 4.95 Å across 57 C α atoms (Fig. 3). Compared with aqueous conditions, SDS micelles may better mimic the bacterial membrane. Further elucidation of the crystal structure of plantacyclin B21AG in membrane-mimicking SDS micelle solvent system may provide a more direct comparison with the NMR solution structure of acidocin B and hence provide clues to the structural changes that occur in bacteriocin family II proteins upon membrane interaction.

Sequence analysis of circular bacteriocins.
To extend the structural comparison, the sequence of plantacyclin B21AG was aligned against the sequences of 18 bacteriocins that have been previously experimentally characterised as circular. In each case, circularisation had been confirmed structurally or validated via independent experiments including electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) and peptide sequencing [12][13][14][15][16][17][18][19][20][21][22][23][24][26][27][28][29][30] . The details and sequences can be found in Supplementary Table S1. Amino acid sequence alignment of these circular bacteriocins shows two families within the class I circular bacteriocins (Fig. 4), which is consistent with recent phylogenetic analysis 10 . Secondary structure prediction suggests that these circular bacteriocins share considerable structural similarity despite sequence variation both within and between the two families. The results suggest that a sequence length of 69-70 residues gives a five α-helical structure whereas a bacteriocin sequence with fewer than 69 residues gives a four helical structure. Jpred secondary structure prediction 57 matched the experimentally determined secondary structure despite the large sequence diversity of the circular bacteriocins.
The position in the sequence of key amino acids such as charged, buried and aromatic residues appears to be conserved across the circular bacteriocin, which may hint at a key role for these residues in the protein antimicrobial activity. Specifically, the cationic residues located in helix α3 and α4 (helix α4 and α5 in the case of enterocin AS-48) in family I are predominantly but not exclusively lysine. In family II, histidine at position 11 and lysine at position 19 are highly conserved. These residues generally appear in polycationic clusters within the first, second, fourth/fifth α-helices. Upon N-to C-termini ligation, these cationic residues are brought into close proximity.
Anionic residues in the circular bacteriocins are generally conserved, with almost all being found in close proximity to the membrane-interacting polycationic region and aromatic residues (Fig. 4). This matches the pattern observed with the structurally determined circular bacteriocins (Fig. 2, right panel). The N-C linkage point is buried in the hydrophobic core in the majority of experimentally determined structures of circular bacteriocins. In family I, the N-and C-terminal residues are generally conserved, sharing common characteristics. For example, the N terminus of family I utilises most commonly a leucine, then valine and methionine. These are all hydrophobic residues which are similar in size (117-149 Da). The C terminus consists of an aromatic residue, either a tryptophan, tyrosine or phenylalanine. Carnocyclin A and garvicin ML are exceptions to this pattern, having a leucine and alanine, respectively at the C-terminus. Instead, a conserved aromatic residue is present in the post-ligation site for carnocyclin A and in the pre-ligation site in garvicin ML. The termini of family II are also highly conserved in both sequence and residue characteristics. The N-terminal residue is often isoleucine, or an aromatic residue, or a valine while the C terminus is an alanine in every case.
Aromatic residues are also highly conserved in family I and II and in addition to the C terminus of almost every family I sequence are found near the termini of predicted or confirmed α-helices. Tryptophan, phenylalanine and tyrosine that flank transmembrane-associated helices in other proteins are thought to facilitate membrane penetration 58,59 . This may also be the case for the circular bacteriocin sequences (Fig. 4). The presence of conserved aromatic residues near α-helices in circular bacteriocins may suggest they assist in cell membrane permeation and pore formation. For example, Trp 24 is essential for the biological activity of AS-48, and is located in a hydrophobic region that interacts with the membrane 60 . Tryptophan has a preference for the interfacial Figure 4. Phylogenetic tree showing the evolutionary relationship between the mature circular bacteriocin sequences, and their aligned sequences. Blue shows cationic residues. Red shows anionic residues. Purple shows aromatic residues. Dark grey highlighting shows α-helices confirmed via structural information. Light grey highlighting shows α-helices predicted by sequence alignment and secondary structure prediction. Underlining shows residues buried in protein core in experimentally determined structures. A. Sequence length of each protein is shown in "residues" column and number of known or predicted α-helices is shown in column labelled α-helices.

Scientific Reports
| (2020) 10:17398 | https://doi.org/10.1038/s41598-020-74332-1 www.nature.com/scientificreports/ region of lipid bilayers and allows penetration of them 61 . Trp 24 is essential for antimicrobial killing activity of gramicidin 62 , specifically for channel formation and conductance 63 and altering energy profiles for ion permeation through long-range electrostatic interactions 59 . Tyrosine and phenylalanine can also have a similar effect 64 , with phenylalanine increasing membrane permeability of proteins 65 .

Mutagenesis of key residues in plantacyclin B21AG.
To confirm the importance of specific residues, we performed site-directed mutagenesis on aromatic and cationic residues within the mature sequence of plantacyclin B21AG. These were non-synonymously substituted with alanine (Ala) to test their importance for antimicrobial activity. Figure 5 shows the location of the three altered residues, Phe 8 , Lys 19 and Trp 45 . Other Ala substitution mutants (Arg 6 and His 11 ) expression plasmids were constructed, however despite multiple attempts to transform the DNA into L. plantarum WCFS1, no transformants were recovered. This could be partly due to bacteriocin toxicity to recombinant host. In general, transformation into L. plantarum WCFS1 was inefficient, requiring many attempts for each construct. The MIC assay results (Table 3) showed plantacyclin B21AG-WT had a value of at least 5.2 ng/µL against indicator strain L. plantarum A6, indicating potent antimicrobial activity against a strain closely related to the bacteriocin producer. By contrast the MIC values of each variant increased, indicating that the antimicrobial activity is reduced. The aromatic residues Phe 8 and Trp 45 appeared to be important for MIC activity; when replaced with Ala mutants the MIC activity was reduced by 48 and 32-fold, respectively. Mutation of aromatic   60 , supporting the notion of a similar role for these residues, despite distinct phylogenetic divergence (Fig. 4). Interestingly, replacement of Lys 19 by Ala had the greatest impact on activity, reducing the antimicrobial activity by 128-fold: 667 ng/µL was required to inhibit growth of the indicator strain as no turbidity was observed in the overnight culture, compared to 5.2 ng/µL for the wild type. The control we used was the untransformed L. plantarum WCFS1 with 10 mM ammonium acetate, which demonstrated no MIC. This control confirms that the inhibitory activity was due to the respective bacteriocins alone. These MIC results identify an important functional role for Phe 8 , Trp 45 and Lys 19 three highly conserved residues identified in the sequence analysis of circular bacteriocins (Fig. 4). The MIC results are in agreement with previous computational inferences that conserved aromatic and cationic residues are important for antimicrobial activity 10 of plantacyclin B21AG and by extension, other circular bacteriocins which share these features.
The observed masses of the wild type plantacyclin B21AG and its variants are listed in Table 4 and presented in Supplementary Figure S1. Notably, a mass was not identified for the plantacyclin B21AG Lys 19 Ala mutant, despite evidence of antimicrobial activity and multiple attempts using fresh cultures and purifications. This mutant had a reduced antimicrobial activity compared with wild type (128-fold reduction). We suggest that substitution of Lys 19 with Ala may result in reduced stability of plantacyclin B21AG. This may explain why it failed to produce a single discrete peak on MALDI-TOF. We hypothecate that degraded fragments may still be antimicrobially active, accounting for the killing assay. A possible explanation is that the N-C terminal circularisation may be compromised in this mutant leading to increased instability 30,39 .

Conclusions
We report the crystal structure of a circular bacteriocin from the food grade Lactiplantibacillus plantarum B21. Structural comparison of plantacyclin B21AG and other circular bacteriocins confirms that these antimicrobial peptides share a highly conserved core fold and secondary structure composition, despite low sequence identity. In the structures solved in aqueous conditions, the N and C ligation point is buried in the hydrophobic core of the globular structure and positioned within an α helix. Sequence alignment of plantacyclin B21AG with other bacteriocins that have been characterised to be circular revealed highly conserved motifs including polycationic regions predicted to be surface exposed that could be involved in membrane interaction, binding and destabilisation. Proximally located anionic residues could be involved in pore selectivity that drives cation flow out of the target cell. Another common motif was the presence of conserved aromatic residues near the N and C terminal ends of helices 4-5. These could be involved in membrane solubilisation, penetration and channel formation. Through multiple lines of evidence including sequence analysis, structure analysis, site-directed mutagenesis and antimicrobial activity, we demonstrated the importance of these evolutionarily conserved residues. In one case, substitution of residue Lys 19 to Ala resulted in 128-fold reduction of antimicrobial activity compared to the wild type plantacyclin B21AG. A problem encountered was the inefficient transformation efficiency of L. plantarum WCFS1 when transforming pTRKH2 backbone plasmids. Future work could focus on improving the efficiency by utilising different plasmid backbones and optimising the transformation protocol, to enable more rapid testing of other variants. Despite this limitation, we demonstrated that three aromatic and cationic residues have key functional roles in the antimicrobial activity of plantacyclin B21AG. Sequence and structure analysis suggest that several other conserved residues are likely to have key functional roles, these include Arg 6 , His 11 and Asp 22 as well as the C and N terminal residues. Sufficient information now exists for the systematic exploration of structure-function relationships, towards the understanding and creation of new microbial killing activities.

Methods
Expression and purification of plantacyclin B21AG. Lactiplantibacillus plantarum B21 was inoculated in 1 L of de Man, Rogosa & Sharpe (MRS) broth using a 2% inoculum (v/v). The culture was incubated for 16-18 h at 30 °C without shaking. The bacteriocin plantacyclin B21AG was purified from the culture supernatant through a four-step protocol, i.e. concentration of cell free supernatant, extraction with water-saturated butanol, desalting through PD10 and cation exchange Fast Protein Liquid Chromatography (FPLC). After overnight incubation, the culture was pelleted at 10,000×g for 10 min. The resulting cell free supernatant (CFS) was concentrated through a 10 kDa polyethersulfone membrane disc using an Amicon Stirred Cell (Merck Table 4. MALDI-TOF mass determination of plantacyclin B21AG-WT and mutants. Also shown is the expected size and mass differences. N/A not applicable. www.nature.com/scientificreports/ Millipore, Germany) to a final volume of 50 mL. The concentrated CFS was then extracted twice with ½ volume of water saturated butanol. The butanol fraction containing plantacyclin B21AG was dried under a fine stream of nitrogen to remove the butanol. The dried butanol fraction was then redissolved in 20 mM of sodium phosphate buffer (pH 6) and desalted using a NAP10 desalting column prepacked with Sephadex G-25 resin (GE healthcare, USA). Finally, the desalted protein fraction was purified using the Uno S6 prepacked monolith cation exchange column (12 × 53 mm, 6 mL, Bio-Rad, USA) (Supplementary Figure S2). The eluted fraction containing purified bacteriocin was concentrated to 4.6 mg/mL in an Amicon centrifugal filter concentrator with a 3 kDa cutoff membrane (Millipore) in buffer comprising 20 mM sodium phosphate (pH 6) for subsequent protein crystallisation.

L. plantarum WCFS1 Sample
Protein crystallisation and data collection. Protein 66 . Crystals were flash-cooled and stored in liquid nitrogen before transferring to a stream of nitrogen gas at 100 K at the beamline. X-ray diffraction data were collected at a wavelength of 0.9537 Å using an EIGER-16 M detector at distance of 230.028 mm and with 0.1° oscillation and 0.1 s exposure of 0% attenuated beam per frame. 3600 frames of the data set were collected in 36 s. The data were processed and scaled using XDS 67 and Aimless 68 , respectively.
Crystal structure determination and refinement. Attempts to solve the X-ray crystal structure of plantacyclin B21AG by molecular replacement (MR) using the NMR structure of acidocin B did not yield a solution. Instead, the phases for plantacyclin B21AG crystal structure were solved by molecular replacement (MR) method with a short model helix structure of 8 amino acids (polyalanine) as a search model, using the program Phaser in the CCP4 suite 69,70 . The program placed six helices in an asymmetric unit with LLG of 393. The six helical model was used in SHELXE [71][72][73] for density modification and polyala model building. The polyala model (109 residues in two chain) resulting from SHELXE was provided to MR phasing protocol of Auto-Rickshaw 74,75 server (https ://www.embl-hambu rg.de/Auto-Ricks haw) for phase improvement, model building and sequence docking. Within the software pipeline, MR was skipped and the partial model was refined in CNS 76 for B-factor and positional refinement. Further refinement in REFMAC5 77,78 resulted in R/Rfree of 38.7%/40.1%. Density modification was performed in PIRATE 79 and the resulting phases were used in ARP/wARP 80 for automated model building. The above density modification and model building procedure were repeated twice, which resulted in a model containing 112 residues with docked sequence in two chains. The last round of refinement (using REFMAC5) gave R/Rfree of 25.7%/30.5%. At this stage, the model was used for manual rebuilding and refinement. 56 water molecules were included where the mFo-nFc difference electron density showed a peak above 3σ and the modelled water made stereochemically reasonable hydrogen bonds. Final refinement was performed with REFMAC5. A Ramachandran plot showed that 99.1% of the residues were in the preferred regions and 0.9% were in the allowed regions. The quality and geometry of the final structure were evaluated and validated using wwPDB validation system 81 . Figures were prepared using PYMOL 56 . Data collection and refinement statistics are presented in Table 1. The final refined model has been deposited in the Protein Data Bank with the code 6WI6 using wwPDB OneDep system 82 .

Structural analysis. Superposition of Chain A and Chain B of Plantacyclin B21AG was performed using
WinCoot 0.8.9.2 83 . The percentage of solvent exposure of each amino acid residue was calculated using GETAREA 1.0 beta (https ://curie .utmb.edu/getar ea.html) 45 , a web service provided by the Sealy Center for Structural Biology at the University of Texas Medical Branch. The electrostatic potential surface map was calculated using the APBS functionality 84 in the PDB2PQR web server (version 2.1.1) (https ://nbcr-222.ucsd.edu/pdb2p qr_2.1.1/) 85 and the figures generated using Chimera 1.14 86 . The isoelectric focusing point of the proteins was computed using the Compute pI/MW tool in ExPASy (https ://web.expas y.org/compu te_pi/). Structural alignment between circular bacteriocins was performed using the Align function in PyMOL 56 .
The Site-directed mutagenesis. Plasmid construct pCycB21 43 was used as a basis for mutagenesis studies.
998 bp inserts containing site-directed mutations were generated via DNA synthesis (Biomatik), flanked with BamHI/PciI restriction sites and 6 base pair (bp) protection bases. These were subcloned into pCycB21, replacing the wild type (WT) sequence. The details of each construct are found in Table 5.
Transformation into plantacyclin B21AG-deficient strain. As previously shown, Lactiplantibacillus plantarum WCFS1 shares 99% genome identity to original plantacyclin B21AG producer L. plantarum B21 89 . The generated constructs were transformed into Lactiplantibacillus plantarum WCFS1 (ATCC BAA-793) 43 via electroporation 90 with some alterations. Briefly, 0.1 mL of an overnight culture was inoculated into 5 mL MRS media (Oxoid) containing 1% glycine and 0.75 M sorbitol. Cells were grown at 37 °C until mid-exponential phase (OD 600 of 1.5-3) then centrifuged at 4000×g for 5 min at 4 °C. From this point on, cells were kept on ice and centrifuged as above. Cells were then washed twice with a 10:1 volume of transformation solution containing 950 mM sucrose and 3.5 mM MgCl 2 . Cells were resuspended in 80 µL of transformation buffer and 500 ng of plasmid DNA was added to cells. This mixture was then added to 0.2 cm cuvette (Bio-Rad) and electroporated with a Gene Pulser (Bio-Rad) with the following conditions: 2 kV, 25 μF and 400 Ω. If the time constant was ≥ 10, another 500 ng of DNA was added, and the electrical pulse repeated. 1 mL of MRS containing 100 mM MgCl 2 and 0.5 M sucrose was used to quickly rescue cells from cuvette. The mixture was added to 1.5 mL microtubes and cells were incubated at 37 °C for 3 h, then 200 µL were spread onto MRS agar plates containing 15 µg/mL of erythromycin (Sigma-Aldrich). Plates were grown at 37 °C for 48 h and colonies screened via erythromycin resistance phenotype and Polymerase Chain Reaction (PCR). Sanger sequencing was performed on the PCR products using both primers to confirm the transformants, at the Griffith University DNA Sequencing Facility, QLD, Australia. Partial-purification of L. plantarum WCFS1-derived bacteriocins. After L. plantarum WCFS1 transformants had been confirmed, they were grown for 24 h in 100 mL MRS cultures containing 15 µg/mL erythromycin. Untransformed L. plantarum WCFS1 cells were also grown overnight in MRS without antibiotics to serve as a negative control. Cells were centrifuged at 8000×g for 10 min, then supernatant concentrated to 2 mL using 3 kDa Amicon Ultra-15 Centrifugal Filter Units (Merck). Supernatant was then washed 3 times with 15 mL 10 mM ammonium acetate to remove residual erythromycin. Concentrated supernatant was then n-butanol extracted as above and then removed in a rotary evaporator. The dried fraction was then resuspended in 10 mM ammonium acetate. Concentration of the bacteriocin from the partial purification was calculated using the extinction coefficient 91 and 280 nm absorbance value using the ProtParam calculator https ://www. protp aram.net/index .html. Concentration was also evaluated with a Bradford assay, using bovine serum albumin as a standard to increase confidence in values generated (data not shown).