Identification and three-dimensional structure of

carnobacteriocin XY, a class IIb bacteriocin produced by

Carnobacteria
Jeella Z. Acedo1, Kaitlyn M. Towle1, Christopher T. Lohans1, Mark Miskolzie1, Ryan T. McKay1,
Thomas A. Doerksen2, John C. Vederas1 and Leah A. Martin-Visscher2
1 Department of Chemistry, University of Alberta, Edmonton, AB, Canada
2 Department of Chemistry, The King’s University, Edmonton, AB, Canada

Correspondence
L. A. Martin-Visscher, Department of
Chemistry, The King’s University, 9125 50th
Street, Edmonton, AB, Canada, T6B 2H3
Fax: +1 780 465 3534
Tel: +1 780 465 3500 ext. 8169
E-mail: leah.martin-visscher@kingsu.ca
(Received 1 March 2017, revised 3 April
2017, accepted 5 April 2017, available online
27 April 2017)
doi:10.1002/1873-3468.12648
In this study, we report that CbnX (33 residues) and CbnY (29 residues) com-
prise a class IIb (two-component) bacteriocin in Carnobacteria. Individually,
CbnX and CbnY are inactive, but together act synergistically to exert a nar-
row spectrum of activity. The structures of CbnX and CbnY in structure-
inducing conditions were determined and strongly resemble other class IIb
bacteriocins (i.e., LcnG, PlnEF, PlnJK). CbnX has an extended, amphipathic
a-helix and a flexible C terminus. CbnY has two a-helices (one hydrophobic,
one amphipathic) connected by a short loop and a cationic C terminus. CbnX
and CbnY do not appear to interact directly and likely require a membrane-
bound receptor to facilitate formation of the bacteriocin complex. This is the
first class IIb bacteriocin reported for Carnobacteria.

Edited by Stuart Ferguson Keywords: antimicrobial peptide; bacteriocin; Carnobacteria; class IIb;
lactic acid bacteria; NMR solution structure

Bacteriocins are ribosomally synthesized antimicrobial
peptides that are produced naturally by bacteria as a
means of self-defense [1–4]. For decades, bacteriocins
produced by lactic acid bacteria (LAB) have been
extensively studied [3,4]. These small, heat-stable pep-
tides display highly selective and potent activity
against other bacteria, including numerous food spoi-
lage and pathogenic bacteria. Since LAB and their
products are generally regarded as safe (GRAS), there
is tremendous potential for the use of these bacteri-
ocins in the food safety industry, and in human and
veterinary medicine [1–3,5].
Bacteriocins produced by LAB are grouped into two
families [1]. Class I bacteriocins (lantibiotics) are char-
acterized by the presence of post-translationally modi-
fied amino acids, such as lanthionine and
methyllanthionine [6,7]. The class II bacteriocins are
further divided into four subclasses and do not contain
modified amino acids [1]. Class IIa bacteriocins are
characterized by their antilisterial activity and high
degree of sequence homology in their N termini,
including the YGNGV consensus sequence and a
disulfide bridge [2,4]. Class IIb bacteriocins require the
combined action of two unique peptides to elicit full
activity [8,9]. Class IIc contains the highly stable circu-
lar peptides, characterized by a peptide bond linking
the N and C termini [7,10]. The class IId (miscella-
neous) bacteriocins are those that do not readily fit
into any of the other classes. These peptides are typi-
cally linear and unmodified, and include the sec-depen-
dent and leaderless bacteriocins [11].
Most bacteriocins from LAB are initially produced
as inactive precursors, with an N-terminal leader
sequence that is cleaved from the bacteriocin during

Abbreviations
CD, Circular dichroism; GRAS, generally regarded as safe; LAB, lactic acid bacteria; NOE, nuclear Overhauser effect; RMSD, root-mean-
square deviation; TFE, trifluoroethanol.

FEBS Letters 591 (2017) 1349–1359 ª 2017 Federation of European Biochemical Societies 1349
Solution structure of the class IIb bacteriocin CbnXY
export from the cell. Many of the class II bacteriocins
have leader sequences ending with a characteristic dou-
ble glycine-motif (GG-motif) [1]. In bacterial genomes,
the presence of orfs encoding small peptides that con-
tain GG-type leader sequences has been used as the
basis to predict bacteriocin-like substances. In 1997, it
was reported that the cbnXY gene cluster found in
Carnobacterium piscicola LV17B likely encoded class
II bacteriocin precursors with GG-type leaders [12].
Indeed, one of the predicted bacteriocins, carnobacteri-
ocin X (CbnX), was subsequently isolated from the
culture supernatant of Carnobacterium maltaromaticum
C2 [13]. Using tandem mass spectrometry and genetic
analysis, it was confirmed that CbnX is a 33-amino
acid peptide, initially produced as a 51-amino acid pre-
cursor as encoded by the cbnX structural gene.
When CbnX was first isolated and characterized, it
was reported to be a class IId bacteriocin [13].
Recently, our group has elucidated the structures of
several leaderless class IId bacteriocins, namely ente-
rocins 7A and 7B [14], lacticin Q [15], and aureocin
A53 (a non-LAB bacteriocin) [15]. Interestingly, our
studies have shown that these broad-spectrum antimi-
crobial peptides share a common structural motif [15].
CbnX is shorter than these class IId bacteriocins and
is not leaderless, therefore we were interested in exam-
ining its structure and function. However, our current
work suggests that CbnX is actually one peptide in a
two-component (class IIb) bacteriocin. Our hypothesis
is based on the following observations. First, heterolo-
gous expression of CbnX failed to yield active bacteri-
ocin. Second, in both C. piscicola LV17B and
C. maltaromaticum C2, the cbnX gene is located beside
the cbnY gene, which also encodes a putative bacteri-
ocin-like substance with a GG-type leader (Fig. 1). It
has been demonstrated that these genes are cotran-
scribed, which would suggest that CbnX and CbnY
are a two-component bacteriocin [12,16]. Third, as
illustrated in Fig. 1, the primary structures of CbnX
and CbnY contain GXXXG motifs, which are charac-
teristic of the class IIb bacteriocins [9]. CbnY has three
additional GXXXG-like motifs, in which either alanine
or serine residues replace a glycine.
Fig. 1. Primary structures of CbnX and CbnY. The sequence of the entire precursor peptide is provided and the site of cleavage between
the leader and mature bacteriocin is indicated with a black arrow. The GXXXG motifs present in CbnX and CbnY are indicated with red lines
and the glycines are in bold. Additional GXXXG-like motifs in CbnY are indicated with green lines.
1350
J. Z. Acedo et al.
Herein, we report our studies confirming that CbnX
and CbnY comprise a new member of the class IIb
bacteriocins. We have also elucidated the three-dimen-
sional NMR structures of these peptides in structure-
inducing conditions. To the best of our knowledge,
CbnXY is the first confirmed class IIb bacteriocin
found in Carnobacteria.
Materials and methods
Unlabeled CbnX and CbnY
The CbnX and CbnY peptides used for activity testing, cir-
cular dichroism (CD) and interaction studies (described in
Supporting Information) were synthesized and purified to
> 98% purity by GenScript (Piscataway, NJ, USA). Amino
acid sequences for the mature CbnX and CbnY peptides
are listed in Fig. 1. The identity of the peptides was con-
firmed by mass spectrometry.
Activity assays of CbnX and CbnY
CbnX and CbnY were dissolved in sterile buffer (20 mM
sodium phosphate, pH 7.5) to a concentration of 1 mM and
a series of twofold dilutions were prepared. The activity of
CbnX and CbnY alone and in combination was assessed
using spot-on-lawn assays. Carnobacterium divergens LV13
and C. maltaromaticum A9b
were used as indicator
organisms, while C. piscicola LV17B and C. maltaro-
maticum C2 were used as negative controls. All Carnobacte-
ria strains were grown at 25 °C in brain heart infusion
broth. Molten soft agar (0.75% agar, 10 mL) was inocu-
lated (1% v/v) with an overnight culture of the strain to be
tested and overlaid onto an agar plate. To test the individ-
ual and synergistic activity of CbnX and CbnY, 10 lL of
each peptide (equimolar) was spotted such that the spots
for CbnX and CbnY overlapped, or the samples were
cospotted. In either case, the first spot was allowed to dry
before the second spot was applied. Plates were incubated
overnight at 25 °C and examined for zones of clearing.
The combined activity of CbnX and CbnY was further
quantified using a microtiter plate assay. Briefly, a 1 mM
stock solution consisting of CbnX and CbnY (equimolar,
in 20 mM sodium phosphate buffer, pH 6.9) was prepared,
FEBS Letters 591 (2017) 1349–1359 ª 2017 Federation of European Biochemical Societies
J. Z. Acedo et al.
aliquoted into a 96-well plate, and diluted to 125 lM with all-
purpose tween (APT) broth. From this, a series of twofold
dilutions were prepared. C. divergens LV13 and C. maltaro-
maticum A9b
were grown overnight in APT broth at 25 °C,
and added into the wells. Dilution of the indicators gave an
optical density (OD600) of ~ 0.06 (McFarland standard 0.5).
The plates were incubated at 25 °C and OD600 measurements
were taken every 10 min for 10 h using a plate reader (Spec-
traMax i3x; Molecular Devices, Sunnyvale, CA, USA). The
minimum inhibitory concentration (MIC) was defined as the
total concentration of CbnXY that inhibited growth by 50%,
as determined by comparing the OD600 of sample wells to
control wells (no bacteriocin) once bacteria reached station-
ary growth phase. The reported MIC values are the average
of two measurements.
Circular dichroism (CD)
Spectra were recorded with an OLIS DSM 17CD spec-
trophotometer (Bogart, GA) using a thermally controlled
quartz cell with a 0.02-cm path length. Data were collected
every 1 nm, from 185 to 260 nm, at 27 °C. Peptide samples
were prepared in either aqueous buffer (20 mM sodium
phosphate pH 6.0) or 50% trifluoroethanol (TFE) in aque-
ous buffer at a concentration of 0.25 mgmL
1 for CbnX
and 0.22 mgmL
1 for CbnY. Results are the average of
five scans and are expressed in units of molar ellipticity
(deg cm2dmol
1). Baseline spectra of the appropriate sol-
vent systems were subtracted from sample spectra prior to
calculating molar ellipticity. The a-helical content of the
peptides was determined by the single point method using
the molar ellipticity at 222 nm, [h]222, according to the fol-
lowing expression:
% helicity ¼ ½h222 =½
40 000ð1
2:5=nÞ  100%;
where n represents the number of peptide bonds [17].
Construction of expression vectors for CbnX and
CbnY
Gene sequences encoding the mature CbnX and CbnY pep-
tides were codon-optimized for Escherichia coli expression
and purchased from BioBasic Inc. (Markham, ON,
Canada). The genes were amplified through PCR using pri-
mers CTL72 (50 -TGGGGTTGGAAAGAAGTGGTTCA
GAATGG-30 ) and CTL73 (50 -TTAGCCAAACGGCAGC
GGCAC-30 ) for cbnX, and CTL76 (50 -TCTGCAATCCT
GGCTATCACTCTGG-30 ) and CTL98 (50 -TTACTTTTT
GCGACGGTCGTTAATGGC-30 ) for cbnY, and then
cloned into the pET SUMO (small ubiquitin-like modifier)
expression vector according to the manufacturer’s instruc-
tions (Invitrogen). Plasmids from successful transformants
were purified using a plasmid DNA mini-prep kit (Qiagen)
and sequenced to confirm that the genes were correct and
FEBS Letters 591 (2017) 1349–1359 ª 2017 Federation of European Biochemical Societies
Solution structure of the class IIb bacteriocin CbnXY
in frame with the His-tagged SUMO fusion protein. The
resulting pET SUMO-CbnX and pET SUMO-CbnY plas-
mids were stored at
20 °C.
Expression of [13C,15N]-labeled fusion proteins
E. coli BL21(DE3) cells (Invitrogen) were transformed sepa-
rately with the pET SUMO-CbnX and pET SUMO-CbnY
plasmids according to manufacturer’s protocols. Transfor-
mants were used to prepare overnight cultures, which were
grown at 37 °C with shaking (225 rpm) in [13C,15N]-labeled
BioExpress Cell Growth Media (Cambridge Isotope Labora-
tories, Inc., Tewksbury, MA, USA) containing 50 lgmL
1
kanamycin. For large-scale expression of the fusion proteins,
either 2 L (for SUMO-CbnX) or 1 L (for SUMO-CbnY) of
the [13C,15N]-labeled media containing 50 lgmL
1 kanamy-
cin was inoculated (1% v/v) with the appropriate overnight
culture. Cultures were grown at 37 °C with shaking
(225 rpm) to an OD600 of 0.8–1.0, placed on ice for 20 min,
and then induced by the addition of 0.5 mM isopropyl-b-D-1-
thiogalactopyranoside. Cultures were incubated for an addi-
tional 24 h at 25 °C with shaking, (225 rpm) after which cells
were harvested (5000 g, 15 min, 4 °C), and stored at
80 °C.
Purification and cleavage of [13C,15N]-labeled
fusion proteins
Cell pellets were resuspended in 30 mL of lysis buffer [50 mM
NaH2PO4 (pH 8.0), 500 mM of NaCl, 10 mM of imidazole,
1% glycerol], and lysed by passing through a Constant Sys-
tems Cell Disruptor, model TS (Constant Systems, Ltd.,
Daventry, Northans, UK) at 20 kpsi. The lysate was cen-
trifuged (15 000 g, 30 min, 4 °C) and 1–2 mL of Ni-nitrilo-
triacetic acid agarose resin (Qiagen) was added to the
supernatant. The mixtures were shaken (50 rpm) for 1 h at
8 °C, transferred to empty columns equipped with frits, and
packed by gravity. The resin was washed with buffer [50 mM
NaH2PO4 (pH 8.0), 500 mM NaCl, 25 mM imidazole], after
which the fusion proteins were eluted by increasing the
amount of imidazole in the buffer (up to 1 M). Fractions con-
taining the desired proteins, as monitored by SDS/PAGE,
were pooled and diluted to obtain a final concentration of
50 mM imidazole and 150 mM of NaCl. With the addition of
stock solutions, the buffer was adjusted to 50 mM Tris-HCl
(pH 8.0), 0.2% IGEPAL CA-360 (Sigma-Aldrich, Oakville,
ON, Canada), and 1 mM dithiothreitol. To cleave the fusion
proteins, His-tagged SUMO protease (McLab, South San
Francisco, CA, USA) was added to the solutions at a ratio of
50 lL protease (100 UlL
1) per 1 L batch of cell culture,
and incubated at 25 °C for 20 h. SUMO-CbnX and SUMO-
CbnY were cleaved with ~ 70% and ~ 100% efficiency,
respectively, as estimated by SDS/PAGE. Neither increasing
the amount of protease nor lengthening the incubation time
resulted in additional cleavage for SUMO-CbnX.
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Solution structure of the class IIb bacteriocin CbnXY
Purification of [13C,15N]-CbnX and [13C,15N]-CbnY
To remove the His-tagged SUMO protease and cleaved
SUMO component of the fusion protein, the cleavage mix-
tures were passed through a column containing 1 mL Ni-
nitrilotriacetic acid agarose resin (Qiagen). CbnX and CbnY
were subsequently purified from the flow-through via reverse-
phase high-performance liquid chromatography (RP-HPLC)
using a C18(2) LUNA AXIA column (5 lm particle size,
100 A  pore size, 21.2 mm 9 250 mm). Water and acetoni-
trile, both containing 0.1% trifluoroacetic acid, were used as
solvents at a flow rate of 8 mLmin
1, and detection at
220 nm. After 10 min at 30% acetonitrile, the gradient was
increased to 95% acetonitrile over 30 min. CbnX eluted at
27 min, while CbnY eluted at 25 min. Fractions containing
CbnX or CbnY were pooled, concentrated under vacuum,
lyophilized, and stored at
20 °C. Approximately 1–1.5 mg
each of [13C,15N]-CbnX and [13C,15N]-CbnY were obtained.
NMR spectroscopy
[13C,15N]-CbnX and [13C,15N]-CbnY were each dissolved in
350 lL 1 : 1 2,2,2-trifluoroethanol-d3:water (pH 6) to a
final concentration of ~ 1 mM. The reference standard 4,4-
dimethyl-4-silapentane-1-sulfonic acid was added at 0.01%
w/v, and the resulting solution was placed into a 5 mm
D2O-matched Shigemi NMR tube (Shigemi Inc., Allison
Park, PA, USA). NMR experiments were performed at
27 °C using a Varian VNMRS 700 MHz spectrometer and
a triple resonance HCN-coldprobe with z-axis pulsed-field
gradients, and VNMRJ 4.2A as host control. The following
two-dimensional (2D) experiments were performed: 1H,1H–-
TOCSY, 1H,1H–NOESY, 1H,15N–HSQC, and 1H,13C–
HSQC. Three-dimensional (3D) experiments included
1 Antimicrobial activity of CbnX and CbnY
H,15N–TOCSY-HSQC, 1
H,15N–NOESY-HSQC, and
HCCH-TOCSY. Experimental parameters are listed in the
Supporting Information (Table S1). For the homonuclear
Table 1. Structure calculation statistics for CbnX and CbnY.
Total NOE peak assignments
Short-range, |i
j| ≤ 1
Medium-range, 1 < |i
j| < 5
Long-range, |i
j| ≥ 5
Average target function value (A2 )
Dihedral angle restraints (TALOS)
RMSD ( A)
Backbone atoms
Heavy atoms
Ramachandran plot (%)
Φ/Ψ in most favored regions
Φ/Ψ in additionally allowed regions
Φ/Ψ in generously allowed regions
Φ/Ψ in disallowed regions
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J. Z. Acedo et al.
2D experiments, water suppression was achieved either by
presaturation (~ 80 Hz gammaB1 induced field) during the
relaxation delay or via water gradient tailored excitation
[18]. NMRPipe [19] and NMRView [20] were used to pro-
cess and analyze the datasets. Chemical shift assignments
are listed in the Supporting Information (Tables S2–S5) and
have been deposited in the Biological Magnetic Resonance
Data Bank with accession numbers 30236 and 30235 for
CbnX and CbnY, respectively.
Structure calculations
The structures of CbnX and CbnY were calculated with
CYANA 2.1 [21] using a combination of automatically and
manually assigned nuclear Overhauser effect (NOE) restraints
from the 1H,1H–NOESY and 1H,15N–NOESY-HSQC spec-
tra, and angle restraints obtained from TALOS [22]. Each
calculation involved seven cycles with 10 000 steps per cycle.
Simulated annealing calculated 100 conformers and the 20
lowest energy conformers were used for further analysis. The
structure calculation statistics, which include NOE peak
count, target function values, root-mean-square deviation
(RMSD), and Ramachandran plot (Fig. S4) data, are sum-
marized in Table 1. There were no structural violations
encountered for either CbnX or CbnY. PyMOL [23] was used
to generate figures and ADAPTIVE POISSON-BOLTZMANN Software
(http://www.poissonboltzmann.org/) [24] was employed to
generate electrostatic surface maps. Coordinates for CbnX
and CbnY were deposited in the Protein Data Bank with
accession numbers 5UJR and 5UJQ, respectively.
Results
The activity of synthetic CbnX and CbnY, individu-
ally and in combination, was evaluated against two
CbnX CbnY
344 333
235 208
109 118
0 7
4.50 E-02  4.06 E-03 8.94 E-03  9.00 E-03
38 46
Residues 2–26 Residues 1–29
0.82  0.36 0.68  0.18
1.37  0.35 1.30  0.22
91.7 82.8
8.3 17.0
0.0 0.2
0.0 0.0
FEBS Letters 591 (2017) 1349–1359 ª 2017 Federation of European Biochemical Societies
J. Z. Acedo et al.
indicator strains (C. divergens LV13 and C. maltaro-
maticum A9b
) using spot-on-lawn assays. Individu-
ally, neither CbnX nor CbnY displayed any
detectable activity even at concentrations as high as
500 lM. However, when CbnX and CbnY were either
cospotted or overlapped, zones of inhibition were
observed at individual peptide concentrations as low
as 4 lM for C. divergens LV13 and 8 lM for C. mal-
taromaticum A9b
(Fig. 2). As a negative control,
CbnX and CbnY were tested against the producer
organisms C. piscicola LV17B and C. maltaromaticum
C2, and no activity was observed confirming that the
producer organisms are immune to CbnXY. Using a
microtitre plate assay, it was determined that the
MIC of CbnXY toward C. divergens LV13 and
C. maltaromaticum A9b
was 15.6 and 31.25 lM,
respectively.
CD studies of CbnX and CbnY
The CD spectra of CbnX and CbnY in aqueous and
structure-inducing conditions were obtained (Fig. 3)
and the helical content of the peptides was predicted
using the single point method [17]. In aqueous condi-
tions, CbnX was unstructured (~ 4% helicity),
whereas CbnY appeared to have some secondary
structure (~ 39% helicity). When exposed to TFE,
Fig. 2. Spot-on-lawn assay showing the synergistic effect of CbnX
and CbnY at different concentrations (indicator: C. maltaromaticum
A9b
). Antimicrobial activity, evidenced by zones of clearing, was
only observed in regions where the peptides overlapped.
FEBS Letters 591 (2017) 1349–1359 ª 2017 Federation of European Biochemical Societies
Solution structure of the class IIb bacteriocin CbnXY
both peptides became highly structured, with a-helical
contents of 78% and 81% for CbnX and CbnY,
respectively.
Production of [13C,15N]-labeled CbnX and CbnY
Isotopically labeled CbnX and CbnY peptides were
prepared by overexpressing the peptides as SUMO
fusion proteins in [13C,15N]-enriched media. After
cleaving the fusion proteins, CbnX and CbnY were
purified by RP-HPLC. Using mass spectrometry, the
incorporation of 13C and 15N isotopes was deter-
mined to be > 95% (see Supporting Informa-
tion, Fig. S2). In total, 1–1.5 mg of each peptide
was obtained and used for subsequent homonu-
clear and heteronuclear multidimensional NMR
experiments.
3D NMR solution structure of CbnX
Sequential assignment of chemical shifts was accom-
plished using a suite of 2D and 3D experiments,
which include 1H,1H–TOCSY, 1H,1H–NOESY,
1
H,15N–HSQC, 1H,13C–HSQC, 3D–1H,15N–TOCSY-
HSQC, 3D–1H,15N–NOESY-HSQC, and HCCH-
TOCSY. CbnX is composed of 33 residues, including
two proline residues. Out of the 31 backbone HN
signals, 30 well-dispersed cross-peaks were observed
in the 15N-HSQC spectrum (Fig. S3A) signifying a
defined structure in solution. The signal for the N-
terminal amino group was not observed, likely due to
rapid exchange with the solvent. The chemical shift
peaklist, distance restraints acquired from the NOE
experiments, and angle restraints from TALOS [22]
were inputted to CYANA 2.1 and used for the struc-
ture calculations. Structural statistics are listed in
Table 1. CbnX is composed of an a-helical chain that
extends from Gly2 to Lys26 and a flexible C terminus.
The ensemble of the 20 lowest energy conformers is
shown in Fig. 4A. For the backbone atoms of the a-
helical region, these structures exhibit an RMSD of
 TALOS predicted an a-helical stretch from
0.82 A.
Gly2 to Val24, which is consistent with the calculated
structure. CbnX contains two consecutive GXXXG
motifs, involving Gly17, Gly21, and Gly25. As illus-
trated in Fig. 4B, these residues are all located on the
same face of the a-helix. Surface analysis showed that
the extended a-helical chain is amphipathic, having
strips of hydrophobic residues on one side and
hydrophilic residues on the other (Fig. 4C). The elec-
trostatic potential surface map (Fig. 4D) reveals that
CbnX has a primarily neutral surface with a few
cationic patches attributed to lysine residues (Lys4,
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Solution structure of the class IIb bacteriocin CbnXY
Fig. 3. CD spectra of (A) CbnX and (B) CbnY in aqueous conditions (+, 20 mM sodium phosphate, pH 6.0) and structure-inducing conditions
(● 50% TFE/aqueous buffer).
Lys19, and Lys26) that are distributed throughout the
chain.
3D NMR solution structure of CbnY
The structure of CbnY was calculated using the same
approach as with CbnX. The 1H,15N-HSQC spectrum
(Fig. S3B) shows all the backbone HN signals except
for the N-terminal amino group. For structure calcu-
lations, 333 NOE restraints were utilized, 208 of
which were short-range, 118 medium-range, and 7
long-range (Table 1). The 20 lowest energy conform-
ers (Fig. 5A) exhibit a backbone RMSD of 0.68 A. 
CbnY is composed of two a-helices in an ‘L’ shape
conformation. Ala2 to Tyr15 (14 residues) comprise
the N-terminal a-helix that is connected by a loop to
the C-terminal a-helix, spanning Met17 to Arg27 (11
residues). The helical sections predicted by TALOS
(Ala2 to Thr13 and Met17 to Arg26) are consistent
with the calculated structure. The GXXXG motif in
CbnY, involving Gly14 and Gly18, is located in the
flexible loop (Fig. 5B). Surface analysis showed that
the N-terminal a-helix is hydrophobic. The C termi-
nus, on the other hand, is composed of an amphi-
pathic a-helix capped with a hydrophilic end that is
composed of four consecutive basic residues
(Fig. 5C). These residues impart a cluster of positive
charge at the C terminus of the peptide, as shown by
the electrostatic potential surface map (Fig. 5D). The
1354
J. Z. Acedo et al.
rest of the solvent-exposed surface appears to be
uncharged.
Discussion
The class IIb bacteriocins produced by LAB require
two complementary peptides to be present in approxi-
mately equal amounts for optimal antimicrobial activ-
ity [8,9]. Since the discovery of lactococcin G (LcnG)
in 1992 [25], the first bacteriocin of this group, at least
15 additional class IIb bacteriocins have been
described [9]. We now report that CbnX and CbnY,
encoded by the cbnXY gene cluster that has been
observed in at least two different strains of Carnobac-
teria [12,13], comprise a new member of the class IIb
bacteriocins. Using synthetically prepared peptide, we
have shown that CbnX and CbnY act synergistically
to exert their activity and that neither peptide is active
individually (Fig. 2). It is likely that the previously
observed activity of CbnX toward C. maltaromaticum
A9b
was due to the presence of undetected CbnY or
minute amounts of another bacteriocin from the pro-
ducer organism [13].
Most class IIb bacteriocins display narrow spectra
of activity and are active at very low concentrations,
typically in the picomolar to nanomolar range [8,9].
Oddly, we found that CbnXY required much higher
concentrations (micromolar range) to be active. We
tested several other strains of Carnobacteria, Listeria
FEBS Letters 591 (2017) 1349–1359 ª 2017 Federation of European Biochemical Societies
J. Z. Acedo et al.
Fig. 4. NMR solution structure of CbnX.
(A) Ensemble of the 20 best structures.
(B) Cartoon drawing showing the GXXXG
motifs. Glycine residues are labeled and
colored green. (C) Hydrophobic surface
plot, where red indicates hydrophobic and
white indicates hydrophilic. (D)
Electrostatic potential map, where blue
and red indicate positive charge and
negative charge, respectively.
and Lactococcus in order to find a more sensitive
strain, but none were found.
Structural analysis of the class IIb bacteriocins
indicates that many of these peptides adopt helical
structures in the presence of membranes or membrane-
mimicking conditions. For example, CD studies have
shown that the individual peptides of LcnG [26,27],
PlnJK [28,29], PlnEF [28,30], and PlnS [31] are ran-
domly coiled in aqueous conditions, but become heli-
cal when exposed to either liposomes, micelles, or the
structure-inducing cosolvent TFE. Similarly, we found
that when CbnX and CbnY were exposed to TFE,
both peptides assumed a high degree of helicity
(Fig. 3).
The helicities of CbnX and CbnY, as predicted from
their CD spectra in 50% TFE, are consistent with the
calculated NMR structures of the peptides. Analysis of
CD data for CbnX indicates that the peptide is 78%
helical, corresponding to ~ 26 out of 33 residues.
Indeed, the NMR structure of CbnX contains a helix
comprised of 25 residues. Likewise, the CD data for
CbnY suggest that the peptide is 81% helical,
FEBS Letters 591 (2017) 1349–1359 ª 2017 Federation of European Biochemical Societies
Solution structure of the class IIb bacteriocin CbnXY
corresponding to ~ 24 out of 29 residues. The calcu-
lated structure of CbnY shows that 25 residues are
involved in helical segments.
The 3D NMR solution structures of CbnX and
CbnY resemble the previously reported structures of
PlnEF [30], PlnJK [29], and LcnG [26]. In particular,
CbnX shares several features with PlnF (from PlnEF),
PlnK (from PlnJK), and LcnG-b (from LcnG): these
peptides contain an amphiphilic a-helix that extends
through the central region of the peptide, on which is
located the GXXXG (or GXXXG-like) motif. It
should be noted that for LcnG-b (in TFE), this helix
is disrupted in two sections, including where the
GXXXG motif is located (residues 18–22). CbnY
shares several features with PlnE, PlnJ, and LcnG-a.
Most notably, these peptides contain two a-helices: a
hydrophobic or amphiphilic a-helix in the N-terminal
half of the peptide and a shorter, hydrophilic, or
amphiphilic a-helix in the C-terminal half. The two
helices are connected by a short, flexible loop, and the
GXXXG motif is located in, or adjacent to the loop.
A visual comparison of the structures of CbnXY,
1355
Solution structure of the class IIb bacteriocin CbnXY
PlnEF, PlnJK, and LcnG is provided in the Support-
ing Information (Fig. S5).
The importance of the GXXXG motifs in the class
IIb bacteriocins has been extensively studied [29,31–
34]. It has been shown that this motif is overrepre-
sented in the transmembrane domains of membrane
proteins [35] and mediates helix–helix interactions of
these domains [35–37]. Thus, for the type IIb bacteri-
ocin, is believed that when GXXXG motifs are located
within an a-helix, they enable the complementary pep-
tides to interact and form a transmembrane helix–helix
structure [8,9]. For LcnG [27], PlnEF [28], and PlnJK
[28], it has also been reported that when complimen-
tary peptides were mixed together in the presence of
liposomes, additional structuring of the peptides was
observed via CD spectroscopy, thus suggesting that
the two peptides directly interact [27,28]. Mutational
analysis of several class IIb bacteriocins including
LcnG [32], PlnJK [29], PlnEF [33], PlnS [31], and bre-
vicin 174A [34], as well as molecular dynamics simula-
tions of PlnS [31] and PlnEF [33,38] have further
1356
J. Z. Acedo et al.
Fig. 5. NMR solution structure of CbnY.
(A) Ensemble of the 20 best structures.
(B) Cartoon drawing showing the flexible
GXXXG motif. Glycine residues are labeled
and colored green. (C) Hydrophobic
surface plot, where red indicates
hydrophobic and white indicates
hydrophilic. (D) Electrostatic potential map,
where blue and red indicate positive
charge and negative charge, respectively.
implicated the importance of specific GXXXG (and
GXXX-like) motifs in the structure and function of
these bacteriocins.
Since CbnX and CbnY contain the characteristic
GXXXG motifs and assume largely helical structures
in structure-inducing conditions, we wanted to explore
if the two peptides directly interact. Therefore, we per-
formed isothermal titration calorimetry studies on
CbnX and CbnY, in the presence of TFE (described in
Supporting Information). However, our results indi-
cated that there is no direct interaction between the
two peptides (Fig. S3). Since it is known that the class
IIb bacteriocins interact with membranes, resulting in
leakage of ions and loss of membrane potential [39–
44], we also looked at the ability of CbnX and CbnY
to interact with vesicles (described in Supporting Infor-
mation). We found that individually, both peptides
permeate the membrane, but no synergistic effect was
observed when the peptides were presented together
(see Supporting Information). Our data, therefore,
strongly suggests that CbnXY requires a specific
FEBS Letters 591 (2017) 1349–1359 ª 2017 Federation of European Biochemical Societies
J. Z. Acedo et al.
receptor molecule to facilitate the formation of an
active bacteriocin complex. Indeed, such receptors
have recently been identified for LcnG [45] and PlnJK
[46]. By generating LcnG-resistant mutants and com-
paring their genomes to the genomes of sensitive
strains, it was discovered that UppP, a membrane pro-
tein involved in peptidoglycan synthesis, is the receptor
for LcnG [45]. In a similar fashion, the receptor for
PlnJK was found to be a putative amino acid trans-
porter [46]. This same strategy could be used to iden-
tify the receptor for CbnXY. However, since CbnXY
is significantly less active, a substantial quantity of
bacteriocin would be needed to generate mutants, and
thus, this approach is currently not feasible.
In conclusion, we have demonstrated that CbnX,
which was previously reported to be a class IId bac-
teriocin, comprises a two-component (class IIb) bac-
teriocin with CbnY as its partner. This is the first
example of a class IIb bacteriocin from Carnobacte-
ria. Furthermore, we elucidated the NMR solution
structures of CbnX and CbnY. The calculated struc-
tures revealed that both peptides share many struc-
tural features with other class IIb bacteriocins.
Future studies will be focused on uncovering addi-
tional proteins that are involved in the processing
and export of CbnXY, as well as conferring immu-
nity to this bacteriocin.
Acknowledgements
We thank Dr Marco van Belkum for his helpful
advice. This work was supported by the Natural
Sciences and Engineering Research Council of Canada
(NSERC) and Alberta Innovates Health Solutions
(AIHS).
Author contributions
LMV and JCV conceived and supervised the study;
JZA, KMT, CTL, LMV, and TAD designed and per-
formed experiments, and analyzed data; RTM and
MM acquired NMR data; LMV, JZA, and KMT
wrote the manuscript; LMV, JZA, KMT, and CTL
made manuscript revisions.
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Supporting information
Additional Supporting Information may be found
online in the supporting information tab for this article:
Doc. S1. Supporting information.
1359