International Journal of

Molecular Sciences
Communication

Characterization of the Novel Leaderless Bacteriocin, Bawcin,
from Bacillus wiedmannii
Zafina Budhwani 1,†,‡ , Jenna T. Buragina 2,†,§ , Jen Lang 2 and Jeella Z. Acedo 1, *
1

2

*
†
‡
§

Department of Chemistry and Physics, Mount Royal University, Calgary, AB T3E 6K6, Canada;
zafina.budhwani@mail.mcgill.ca
Department of Biology, Mount Royal University, Calgary, AB T3E 6K6, Canada;
jenna.buragina@ucalgary.ca (J.T.B.)
Correspondence: jacedo@mtroyal.ca
These authors contributed equally to this work.
Current address: Department of Human Genetics, McGill University, Montreal, QC H3A 0C7, Canada.
Current address: Department of Microbiology and Infectious Diseases, University of Calgary,
Calgary, AB T2N 1N4, Canada.

Abstract: The rise of drug-resistant bacteria is a major threat to public health, highlighting the
urgent need for new antimicrobial compounds and treatments. Bacteriocins, which are ribosomally
synthesized antimicrobial peptides produced by bacteria, hold promise as alternatives to conventional
antibiotics. In this study, we identified and characterized a novel leaderless bacteriocin, bawcin, the
first bacteriocin to be characterized from a Bacillus wiedmannii species. Chemically synthesized and
purified bawcin was shown to be active against a broad range of Gram-positive bacteria, including
foodborne pathogens Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes. Stability
screening revealed that bawcin is stable over a wide range of pH (2.0–10.0), temperature conditions
(25–100 ◦ C), and against the proteases, papain and pepsin. Lastly, three-dimensional structure
homology modeling suggests that bawcin contains a saposin-fold with amphipathic helices and a
highly cationic surface that may be critical for membrane interaction and the subsequent cell death
of its targets. This study provides the foundational understanding of the activity and properties of
bawcin, offering valuable insights into its applications across different antimicrobial uses, including
as a natural preservative in food and livestock industries.
Citation: Budhwani, Z.; Buragina,
J.T.; Lang, J.; Acedo, J.Z.

Keywords: leaderless bacteriocin; antimicrobial; antimicrobial resistance

Characterization of the Novel
Leaderless Bacteriocin, Bawcin, from
Bacillus wiedmannii. Int. J. Mol. Sci.
2023, 24, 16965. https://doi.org/
10.3390/ijms242316965
Academic Editor: Manuel Simões
Received: 11 November 2023
Revised: 26 November 2023
Accepted: 27 November 2023
Published: 30 November 2023

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

1. Introduction
The discovery of antibiotics has saved many lives from infections for decades. Yet, the
continuous emergence of antimicrobial resistance (AMR) is leaving traditional antibiotics
ineffective. This evolution has given rise to various multidrug-resistant strains that significantly threaten human health. The World Health Organization has recognized AMR
as one of the top 10 global public health concerns (https://www.who.int/news-room/
fact-sheets/detail/antimicrobial-resistance; accessed 11 October 2023), where 1.27 million
deaths in 2019 were related to bacterial AMR alone [1]. Hence, the scientific community
has been exploring several avenues to combat AMR, including the discovery of antimicrobial alternatives such as bacteriocins [2]. Bacteriocins are bacteria-derived ribosomally
synthesized antimicrobial peptides that are active against other bacteria, either in the same
species (narrow spectrum) or across genera (broad spectrum) [3]. These compounds are
particularly attractive alternatives to antibiotics because they are natural, can be genetically
modified, and are normally highly stable and non-cytotoxic [4].
Bacteriocins can be grouped based on their general structure and biology; however,
multiple classification schemes have been proposed and continue to change as more information about bacteriocins is discovered. The most current proposed classification includes

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three major classes that are distinguished by heat stability, post-translational modifications,
and size [5]. Each class can be further subdivided according to other structural features,
such as “leaderless” bacteriocins within class II, and specifically classified as class IId [5].
Leaderless bacteriocins are of particular interest because they are not produced with a
leader peptide. Bacteriocins are normally synthesized as inactive precursor peptides consisting of an N-terminal leader peptide and a C-terminal core peptide, where the leader
sequence must be cleaved before the active bacteriocin is released [6]. Leaderless bacteriocins, however, are active upon translation and do not contain a leader sequence [3]. The
leader peptide is known to play an essential role in bacteriocin biosynthesis such as in
conferring producer organism immunity and serving as recognition motifs for biosynthetic
enzymes [6,7]. The absence of a leader peptide in leaderless bacteriocins therefore renders
their immunity and transport mechanisms unconventional and enigmatic [6,7].
Bacteriocin production is a widespread feature of bacterial species [8,9]; however,
identifying bacteriocins from Bacillus species is of particular interest given their high
capacity for antimicrobial compound production [10]. Bacillus bacteria can produce a wide
variety of bacteriocins spanning nearly all bacteriocin classes [10], and new species of this
genus continue to be discovered [11]. Here, we investigated the activity and stability of a
novel leaderless bacteriocin termed bawcin, which is the first bacteriocin to be characterized
from the recently established Bacillus wiedmannii species [11].
2. Results and Discussion
2.1. Bawcin Identification and Biosynthetic Gene Cluster
Our recent genome mining study identified over 400 producer organisms that putatively produce novel leaderless bacteriocins [12]. In this earlier work, an all-by-all BLAST
analysis was performed to compare the amino acid sequences of characterized and putative
leaderless bacteriocins. Results were visualized through a sequence similarity network
wherein the peptides were grouped based on sequence similarity. Bawcin belongs to
Group 1, which is the largest among the 11 groups identified. In detail, Group 1 includes
76 different bacterial strains, mostly Bacillus species, with a few Oceanobacillus, Parageobacillus, and Virgibacillus strains. When we first identified bawcin as a candidate peptide to
isolate and characterize, there were no known members of the said group, and we aimed to
report the first representative member of this family. However, more recently, the bacteriocins NTN-A and NTN-B from Virgibacillus salexigens NT N53, isolated from the deep-sea
floor, were identified and found to belong to the same family as that of bawcin [13]. NTN-A
and NTN-B are 98% identical, while bawcin displays 62.5% sequence identity with NTN-A
and NTN-B (Figure 1a). In contrast to the discovery of NTN-A and NTN-B, which were
isolated from their natural producer organism, we identified bawcin in silico, and it was
synthesized chemically through solid-phase peptide synthesis and HPLC-purified for
subsequent characterization. Its identity was confirmed by mass spectrometry.
B. wiedmannii was recently established as a novel species through an in-depth characterization of 11 B. wiedmannii sp. nov strains, all found in dairy environments between
2005 and 2012 [11]. This species is represented by the type strain FSL W8-0169T , which was
isolated in 2012 from an American dairy powder processing plant raw milk silo [11,14].
Bawcin represents the first bacteriocin to be identified from a B. wiedmannii species. It is
specifically encoded in the genomes of B. wiedmannii strains AFS001911 and AFS057349,
which were isolated from a plant core by AgBiome (Durham, NC, USA) [15,16].
Bawcin was found to be a water-soluble peptide that has 48 amino acids and a molecular weight of 5485.47 Da (Figure 1a; protein accession number WP_098048139.1). Its
biosynthetic gene cluster (BGC) (Figure 1b) has seven genes, four of which are putative
membrane proteins (Table 1). Downstream the bawcin structural gene (bawA) are two genes
(bawB and bawC) that encode proteins with unknown function. BawB is a hypothetical
protein with 94 amino acid residues and three predicted transmembrane domains. BawC is
a soluble protein with 98 residues. BawDEF are putatively involved in bawcin transport
and secretion and consist of 390, 226, and 399 residues, respectively. In particular, BawD

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genes (bawB and bawC) that encode proteins with unknown function. BawB is a hypothetical protein with 94 amino acid residues and three predicted transmembrane domains.
3 of 12
BawC is a soluble protein with 98 residues. BawDEF are putatively involved in bawcin
transport and secretion and consist of 390, 226, and 399 residues, respectively. In particular, BawD is a putative HlyD family efflux transporter with one transmembrane domain,
is a putative HlyD family efflux transporter with one transmembrane domain, BawE is a
BawE is a putative soluble ATP-binding ABC transporter, and BawF is a putative ABC
putative soluble ATP-binding ABC transporter, and BawF is a putative ABC transporter
transporter permease with five membrane-spanning domains. Lastly, BawG is a Yip1 fampermease with five membrane-spanning domains. Lastly, BawG is a Yip1 family membrane
ily membrane protein with 223 residues and five predicted transmembrane domains. Yip1
protein with 223 residues and five predicted transmembrane domains. Yip1 proteins are
proteins are postulated to be involved in transport and were identified to be prevalent in
postulated to be involved in transport and were identified to be prevalent in BGCs of circuBGCs of circular bacteriocins that specifically involve the set of three genes encoding ABC
lar bacteriocins that specifically involve the set of three genes encoding ABC transporter
transporter components, similar to the ones present in the bawcin BGC (i.e., bawDEF) [17].
components, similar to the ones present in the bawcin BGC (i.e., bawDEF) [17].

Figure
Figure 1.
1. (a)
(a)Amino
Aminoacid
acidsequence
sequencealignment
alignmentof
of bawcin,
bawcin, NTN-A,
NTN-A, and
and NTN-B.
NTN-B. Sequences
Sequences were
were
aligned
using
Clustal
Omega
[18].
Conserved,
conservative,
and
semi-conservative
substitutions
are
aligned using Clustal Omega [18]. Conserved, conservative, and semi-conservative substitutions
indicated
by
asterisks,
colons,
and
periods,
respectively.
(b)
Biosynthetic
gene
cluster
of
bawcin
are indicated by asterisks, colons, and periods, respectively. (b) Biosynthetic gene cluster of bawcin
(BawA).
(BawA).The
Thered
redarrow
arrowrefers
refersto
tothe
thebacteriocin
bacteriocinstructural
structuralgene
gene(bawA);
(bawA);gray
grayarrows
arrowsare
aregenes
genes(bawB
(bawB
and bawC) encoding hypothetical proteins of unknown function; green arrows represent genes enand bawC) encoding hypothetical proteins of unknown function; green arrows represent genes
coding putative transporter proteins, specifically, bawD (HlyD family efflux transporter), bawE
encoding putative transporter proteins, specifically, bawD (HlyD family efflux transporter), bawE
(ABC transporter ATP-binding protein), and bawF (ABC transporter permease); and the brown ar(ABC
transporter
ATP-binding
protein),a and
(ABC
transporter permease); and the brown arrow
row indicates
the bawG
gene encoding
Yip1bawF
family
protein.
indicates the bawG gene encoding a Yip1 family protein.
Table 1. Predicted proteins encoded by the bawcin biosynthetic gene cluster 1.
Table 1. Predicted proteins encoded by the bawcin biosynthetic gene cluster 1 .

Protein
Protein
BawA
BawA
BawB
BawB
BawC
BawC
BawD
BawD
BawE
BawE
BawF
BawF
BawG
BawG

Size (aa)
48 Size (aa)
94 48
98 94
98
390 390
226 226
399 399
223 223

TM
0
3
0
1
0
5
5

Identity
TM
Identity
Bawcin (bacteriocin)
0Hypothetical
Bawcin
(bacteriocin)
protein
3
Hypothetical protein
DUF2089 family protein
0
DUF2089 family protein
efflux
transporter
1HlyD family
HlyD
family
efflux transporter
ABC
transporter
ATP-binding
protein protein
0
ABC transporter ATP-binding
5ABC transporter
ABC transporter
permeasepermease
5Yip1 familyYip1
family protein
protein

1 aa, amino acids; TM, transmembrane domains as predicted using the SOSUI program [19].

1

aa, amino acids; TM, transmembrane domains as predicted using the SOSUI program [19].

2.2.Antimicrobial
AntimicrobialActivity
ActivityofofBawcin
Bawcin
2.2.
Bawcinisisactive
activeagainst
againstnine
nineof
ofthe
the fourteen
fourteen bacterial
bacterialspecies
species tested
tested (Table
(Table 2).
2). Given
Given
Bawcin
bawcin
inhibited
the
growth
of
various
bacteria
outside
the
Bacillus
genus,
it
is
considered
bawcin inhibited the growth of various bacteria outside the Bacillus genus, it is considered
broad-spectrumbacteriocin.
bacteriocin.Its
Itsactivity
activityagainst
againstmultiple
multiplenotable
notablefoodborne
foodbornepathogens,
pathogens,
aabroad-spectrum
including
S.
aureus,
B.
cereus,
and
L.
monocytogenes
[20],
demonstrates
bawcin’s
potential
as a
including S. aureus, B. cereus, and L. monocytogenes [20], demonstrates bawcin’s potential
promising
food
biopreservative.
In
addition,
antimicrobials
with
a
broad
activity
range
are
as a promising food biopreservative. In addition, antimicrobials with a broad activity
useful
for
the
treatment
of
infections
in
which
the
causative
bacterial
pathogen
is
unknown.
range are useful for the treatment of infections in which the causative bacterial pathogen
they
may negatively
impact
the human
microbiome
[21].
Bioengineering
[22] and
isHowever,
unknown.
However,
they may
negatively
impact
the human
microbiome
[21]. Bioengicombinatorial
therapy
[23]
can
enhance
the
potency
of
bacteriocins
to
target
species
neering [22] and combinatorial therapy [23] can enhance the potency of bacteriocinsand
to
therefore be used to reduce the impact of broad-spectrum bacteriocins on host microbiota.

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Table 2. Minimum inhibitory concentration (MIC) of bawcin against tested bacteria using spot-onlawn assay 1 .
Indicator Organism

MIC (µM)

Heteroresistance (µM)

Micrococcus luteus (Ward’s 85W 0966)
Lactococcus lactis ssp. lactis (Ward’s 85W 1774)
Lactobacillus delbruekii ssp. bulgaricus (VWR 470179-126)
Bacillus subtilis (Ward’s 85W 1650)
Listeria monocytogenes ATCC 19115
Lactobacillus casei (Ward’s 85W 1682)
Bacillus cereus (Ward’s 85W 0200)
Staphylococcus aureus ATCC 6538
Enterococcus faecalis (Ward’s 85W 1100)
Staphylococcus epidermidis (Ward’s 85W 1747)
Streptococcus pyogenes (Ward’s 85W 1180)
Geobacillus stearothermophilus 1010
Pseudomonas aeruginosa (Ward’s 85W 1173)
Salmonella enterica (Ward’s 85W 1710)

4
4
8
8
8
8
16
16
32
-

4
4
8
8, 16
8, 16
8–32
16–64
16–64
32–128
-

1 -, no activity.

The minimum inhibitory concentrations (MICs) of bawcin are comparable to those of
other broad-spectrum leaderless bacteriocins, specifically those peptides that were tested
using an agar-based assay such as the spot-on-lawn assay employed in this study [24–26].
The spot-on-lawn assay allows for the detection of bacterial heteroresistance (HR). HR
is the phenomenon in which a portion of cells within an otherwise susceptible bacterial
population is resistant to a given antimicrobial [27,28]. HR is distinct from persistence, given
the cells are able to proliferate in the presence of the antimicrobial [29]. Since heteroresistant
populations can grow in the presence of antibiotics, understanding and controlling HR
is necessary to avoid infection treatment failure [27,28]. Bawcin HR was observed for all
susceptible species, although at a minimal level for most organisms (Table 2). In order to
effectively utilize bawcin against these species, techniques to avoid HR should be pursued
in future studies such as through combined application with other antimicrobials that could
enhance bawcin’s activity [30,31].
S. epidermidis, S. pyogenes, G. stearothermophilus, P. aeruginosa, and S. enterica were unaffected by bawcin at all concentrations (Table 2). A lack of activity against P. aeruginosa and
S. enterica was expected given Gram-negative species are typically resistant to bacteriocins
due to their reduced cell wall permeability [3]. Inactivity against these two organisms is
consistent with leaderless bacteriocins produced by other Bacillus species that have been
shown to be bactericidal via inducing bacterial membrane damage [32–34]. Given their
comparable antimicrobial spectra, bawcin may exhibit antimicrobial activity through a
similar mechanism. Further, resistance by the Gram-positive species, S. epidermidis, S.
pyogenes, and G. stearothermophilus, may be due to structural differences, such as a lack
of a receptor necessary for peptide docking to the membrane. Generally, bacteriocins of
this class are believed to initiate pore formation without target receptors [35] and can even
exhibit bactericidal effects without generating membrane pores in target bacteria [36]. However, there are a few leaderless bacteriocins that use receptors, such as LsbB, which only
inhibits the growth of bacteria expressing the YvjB Zn-dependent metallopeptidase [37].
Factors contributing to the need for bacteriocin receptors are poorly understood [38]; yet,
the specificity of bawcin to only certain Gram-positive species suggests a receptor may
be necessary for its antimicrobial activity. Evidently, additional studies are necessary to
establish bawcin’s mode of action and potential target receptor, which will provide insights
into what contributes to bawcin’s selectivity and the additional techniques that are best
suited to enhance its potency and antimicrobial spectrum.

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insights into what contributes to bawcin’s selectivity and the additional techniques that
5 of 12
are best suited to enhance its potency and antimicrobial spectrum.
2.3. pH and Temperature Stability of Bawcin
2.3.Understanding
pH and Temperature
Stability
of Bawcin
bawcin’s
stability
and activity in various chemical and temperature
Understanding
bawcin’s
stability
and activity
in various
chemical
andfor
temperature
environments
is important
as this
information
will guide
the use
of bawcin
various
environments
is important
as this information
will guide
useMIC
of bawcin
various
practical
and industrial
applications.
Bawcin remained
activethe
at the
(8 µM)for
without
practical and industrial
applications.
Bawcin
active
at the
(8(Figure
µM) without
heteroresistance
development
after exposure
to remained
pH 2.0–10.0
against
M.MIC
luteus
2a).
heteroresistance
development
after
exposure
to
pH
2.0–10.0
against
M.
luteus
(Figure
2a).
This activity can be attributed only to bawcin and not from any adverse buffer effects
This
activity
can
be
attributed
only
to
bawcin
and
not
from
any
adverse
buffer
effects
against the indicator organism, as the buffer control did not produce a zone of inhibition.
against
the indicator
as the
buffer control
did not produce
a zone
of inhibition.
The
incubation
time fororganism,
the pH testing
procedure
was optimized
such that
the bacteriocinThe
incubation
time
for
the
pH
testing
procedure
was
optimized
such
that
the
bacteriocinbuffer solutions were incubated for 30, 60, and 120 min. At 30 min after incubation,
the
buffer did
solutions
were incubated
for equilibrated
30, 60, and 120
min.
30 min
after
peptide
not appear
to have fully
with
theAt
buffer.
The
60 incubation,
and 120 minthe
peptide did
notproduced
appear to
have fully
equilibrated
with
the buffer.
The 60
min
incubation
times
consistent
results;
therefore,
60 min
was selected
forand
the 120
assay.
incubation
times
produced
consistent
results;
therefore,
60
min
was
selected
for
the
assay.
Similarly, bawcin also remained active against M. luteus after 1 h exposure to 25–100 °C
◦
Similarly,
bawcin also remained
(Figure
2b), independent
of the pH active
results.against M. luteus after 1 h exposure to 25–100 C
(Figure 2b), independent of the pH results.

Figure
Representativespot-on-lawn
spot-on-lawnassay
assayresults
results for
for bawcin
2.0–10.0)
Figure
2. 2.
Representative
bawcin at
at various
various(a)
(a)pH
pHvalues
values(pH
(pH
2.0–
◦ C) against M. luteus after 1
andand
(b)(b)
temperatures
(25–100
of incubation.
incubation.Bawcin
Bawcinretained
retained
10.0)
temperatures
(25–100
°C) against M. luteus after h of
itsits
activity
at at
8 µM
across
allallconditions
activity
8 µM
across
conditions(circled).
(circled).Respective
Respectiveampicillin
ampicillin(positive
(positivecontrol,
control, +),
+),sterile
sterile wawater
ter(negative
(negativecontrol,
control,-),-),and
andbuffer
buffer(B)
(B)
controls
were
also
spotted.
Assays
were
performed
triplicontrols
were
also
spotted.
Assays
were
performed
in in
triplicates.
cates.

The Bacillus genus has been heavily studied with respect to the bacteriocins and
The Bacillus genus
has been
heavilythey
studied
with respect
to the
bacteriocins
bacteriocin-like
inhibitory
substances
produce
[10,39,40].
This
is because and
this bacgenus
teriocin-like
inhibitoryinsubstances
they produce
[10,39,40].
This isregarded
because as
this
genus
has been implicated
various industrial
processes,
is generally
safe,
easy,has
and
inexpensive
to in
grow;
and industrial
tends to have
broad-range
inhibitory
effectsas[10].
Similar
to the
been
implicated
various
processes,
is generally
regarded
safe,
easy, and
stability testing
results
obtained
forbroad-range
bawcin, several
independent
of bacteriocins
inexpensive
to grow;
andwe
tends
to have
inhibitory
effectsstudies
[10]. Similar
to the
from
Bacillus
species
have
demonstrated
that
these
peptides
are
stable
across
a
wide
pH
stability testing results we obtained for bawcin, several independent studies of bacterioctemperature
range have
[41–43].
insand
from
Bacillus species
demonstrated that these peptides are stable across a wide
applications
of bacteriocins are currently expanding. For example, nisin was
pH andIndustrial
temperature
range [41–43].
firstIndustrial
commercially
introduced
in the 1950s as
a food
preservative
in theFor
UKexample,
[44,45]. Recently,
applications
of bacteriocins
are
currently
expanding.
nisin
bacteriocins
are also being
considered
useasinathe
cosmetic
industry
andUK
human
and
was
first commercially
introduced
in the for
1950s
food
preservative
in the
[44,45].
veterinary
medicine
[46].
Additionally,
there
are
efforts
to
improve
bacteriocins’
antimicroRecently, bacteriocins are also being considered for use in the cosmetic industry and hubialand
efficacy
in various
industrial
applications such
asare
using
encapsulation,
man
veterinary
medicine
[46]. Additionally,
there
efforts
to improve bioengineering,
bacteriocins’
combinatory/complementary
methods,
and
creating
active
antimicrobial
packaging
[47].
antimicrobial efficacy in various industrial applications such as using encapsulation,
bioWith the growing
interest in bacteriocin industrial
the potential
engineering,
combinatory/complementary
methods,applications,
and creatingbawcin
activehas
antimicrobial
to be used
for With
diverse
due toinits
thermal and
chemical
stability, and
further
packaging
[47].
theapplications
growing interest
bacteriocin
industrial
applications,
bawcin
research can be directed towards optimizing conditions for its use in specific applications.

Int. J. Mol. Sci. 2023, 24, 16965

with papain and only at 4× the MIC (with HR) with pepsin (Figure 3). B
crobial activity at all concentrations tested in the presence of trypsin, alp
and proteinase K. Bawcin’s altered antimicrobial activity in combinat
confirms its proteinaceous nature and highlights its potential for food p
6 of 12
antibiotics have been historically overused in food production [48] and
ing demand for naturally derived additives, developing bacteriocins as
2.4. Protease
Stability
of Bawcin area of research. To qualify as a food additive,
has
become
a popular
Bawcin retained its antimicrobial activity at 1×, 2×, and 4× the MIC when combined
(a)
be
non-toxic to human cells and gut microbiota; (b) impair the grow
with papain and only at 4× the MIC (with HR) with pepsin (Figure 3). Bawcin lost antimiinant
bacteria;
retain activity
in presence
the target
food
matrix; (d) and hav
crobial activity
at all (c)
concentrations
tested in the
of trypsin,
alpha-chymotrypsin,
and
proteinase
K.
Bawcin’s
altered
antimicrobial
activity
in
combination
with proteases
different acidity, temperature, and salt levels [49]. Inactivation
of bawc
confirms its proteinaceous nature and highlights its potential for food preservation. Given
proteases,
pepsin,
trypsin,
andinalpha-chymotrypsin,
that
antibiotics have
been historically
overused
food production [48] and theredemonstrates
is an increasing
demand
for
naturally
derived
additives,
developing
bacteriocins
as
food
preservatives
has
be safely degraded within the human gastrointestinal tract upon cons
become a popular area of research. To qualify as a food additive, a bacteriocin must (a) be
tion
to several
pathogens
[20], beneficial
including
non-toxic
to humanfoodborne
cells and gut microbiota;
(b) impair
the growth of bacteria,
food contaminant
bacteria;
(c) retain activity
thesubtilis,
target foodare
matrix;
(d) and have
stability(Table
in different
ssp.
bulgaricus,
andinB.
sensitive
tohigh
bawcin
2). Yet,
acidity, temperature, and salt levels [49]. Inactivation of bawcin by the digestive proteases,
against
these species, has been FDA-approved, and is generally recog
pepsin, trypsin, and alpha-chymotrypsin, demonstrates that bawcin will likely be safely
To
understand
bawcin’s
potential
food
preservation,
future
efforts sh
degraded
within the human
gastrointestinal
tractin
upon
consumption.
In addition
to several
foodborne
pathogens
[20],
beneficial
bacteria,
including
L.
casei,
L.
delbruekii
ssp.
bulgaricus,
ther establishing bawcin’s toxicity and stability in food matrices. Alth
and B. subtilis, are sensitive to bawcin (Table 2). Yet, nisin is also active against these species,
sistant
papain (Figure
3), a protease
found inbawcin’s
meat as a ten
has been to
FDA-approved,
and is generally
recognized commonly
as safe [50]. To understand
potential in food preservation,
shouldof
focus
on further establishing
bawcin’s
considerations
beyondfuture
the efforts
presence
proteolytic
enzymes
include th
toxicity and stability in food matrices. Although bawcin is resistant to papain (Figure 3), a
bawcin
may interact
withasfood
components
and experience
changes
protease commonly
found in meat
a tenderizer
[51], other considerations
beyond the
presence [52].
of proteolytic enzymes include the degree in which bawcin may interact with
charge
food components and experience changes in solubility and charge [52].

Figure 3. Representative spot-on-lawn assay results for bawcin at 32, 16, and 8 µM against M. luteus
Figure
3. Representative spot-on-lawn assay results for bawcin at 32, 16, and 8
following exposure to 1 mg/mL papain and pepsin for 1 h at 37 ◦ C. Bawcin remained active against
following
exposure to 1 mg/mL papain and pepsin for 1 h at 37 °C. Bawcin rem
M. luteus at 8 µM in combination with papain (circle) and partial activity at 32 µM with pepsin
M.
luteus
at 8 (positive
µM incontrol,
combination
with
papain
(circle)
and(P),partial
activity
at
(arrow).
Ampicillin
+), sterile water
(negative
control,
-), protease
and buffer
(B)
controls
were
also
spotted.
Assays
were
performed
in
triplicates.
(arrow). Ampicillin (positive control, +), sterile water (negative control, -), pro
(B) controls
were alsowithin
spotted.
Assayssystem
werebeing
performed
in triplicates.
Despite inactivation
the digestive
advantageous
with respect to

food applications, it is a barrier to developing bawcin for orally administered pharmaceutical use against bacterial infections. However, this issue may be resolved as nanoencapsulaDespite inactivation within the digestive system being advantage
tion delivery systems become more advanced [53] or through genetic modification [54]. For
food
applications,
it is
a residues
barrierwithin
to developing
bawcin
admini
instance,
inserting D-amino
acid
peptide terminal
ends hasfor
beenorally
shown to
reduceuse
protease
sensitivity
[55,56], and
this technique
resulted in athis
relatively
small
loss be
of resol
tical
against
bacterial
infections.
However,
issue
may
antimicrobial activity when implemented in bacteriocins previously [54].

sulation delivery systems become more advanced [53] or through ge
2.5. Structural
Analysis of inserting
Bawcin
[54].
For instance,
ᴅ-amino acid residues within peptide term
The three-dimensional structure of bawcin was predicted using the SWISS-MODEL
shown
to reduce protease sensitivity [55,56], and this technique resu
server [57], visualized using PyMOL 2.0 [58], and shown to consist of four amphipathic
small
loss (Figure
of antimicrobial
activity
when
implemented
in bacteriocins
alpha-helices
4a). In detail, each
helix has
a distinct
strip of hydrophobic
residues
that are oriented toward the core of the structure, while a hydrophilic strip is exposed on
the surface.

2.5. Structural Analysis of Bawcin

Int. J. Mol. Sci. 2023, 24, 16965

The three-dimensional structure of bawcin was predicted using the SWISS-MODE
server [57], visualized using PyMOL 2.0 [58], and shown to consist of four amphipathi
alpha-helices (Figure 4a). In detail, each helix has a distinct strip of hydrophobic residue
7 of 12
that are oriented toward the core of the structure, while a hydrophilic strip is exposed o
the surface.

Figure4. 4.
Predicted
three-dimensional
structure
of bawcin
obtained
the SWISS-MODE
Figure
(a)(a)
Predicted
three-dimensional
structure
of bawcin
obtained
using theusing
SWISS-MODEL
tool
[57]
and
visualized
using
PyMOL
[58].
The
Nand
Ctermini
are
labelled.
A color
gradient colo
tool [57] and visualized using PyMOL [58]. The N- and C- termini are labelled. A gradient
scheme
is
used
where
darker
red
color
indicates
increased
hydrophobicity.
(b)
Hydrophobicity
su
scheme is used where darker red color indicates increased hydrophobicity. (b) Hydrophobicity
face
map
of
bawcin
generated
using
PyMOL.
(c)
Electrostatic
potential
surface
maps
of
bawcin
gen
surface map of bawcin generated using PyMOL. (c) Electrostatic potential surface maps of bawcin
erated using
APBS
online pipeline
pipeline[59].
[59].Cationic
Cationicregions
regionsare
are colore
generated
using
APBSfunctionality
functionalityof
of the
the PDB2PQR
PDB2PQR online
blue. blue.
colored

The
hydrophobicity
surface
mapsmaps
reveal reveal
hydrophobic
patches on
the protein’s
The
hydrophobicity
surface
hydrophobic
patches
on thesurface
protein’s sur
(Figure
4b).
These
structural
features
of
bawcin
are
characteristic
of
the
saposin-like
face (Figure 4b). These structural features of bawcin are characteristic of the saposin-lik
fold structure adopted by many leaderless bacteriocins [60]. Saposin-like folds rely on a
fold structure adopted by many leaderless bacteriocins [60]. Saposin-like folds rely on
combination of hydrophobic and electrostatic interactions to stabilize the structure of the
combination of hydrophobic and electrostatic interactions to stabilize the structure of th
peptide [3]. Although bawcin presents exposed hydrophobic patches, the peptide remains
peptide [3].
presents
exposed
hydrophobic
patches,point
the peptide
hydrophilic
in Although
character asbawcin
it is fully
soluble in
water and
has a high isoelectric
of 10.12. remain
hydrophilic
in
character
as
it
is
fully
soluble
in
water
and
has
a
high
isoelectric
Moreover, the electrostatic surface maps reveal bawcin has a net cationic surface (Figure 4c). point o
10.12. Moreover,
the electrostatic
surface
net cationic
Specifically,
the primary
sequence of bawcin
hasmaps
eight reveal
cationicbawcin
residueshas
(i.e.,a seven
lysine surfac
and
one arginine).
The hydrophobic
and electrostatic
of bawcin,
like residues
with
(Figure
4c). Specifically,
the primary
sequence ofcharacteristics
bawcin has eight
cationic
(i.e
other
leaderless
bacteriocins,
are
thought
to
play
a
key
role
in
the
bacteriocin’s
mode
of
seven lysine and one arginine). The hydrophobic and electrostatic characteristics of baw
action,
particularly
in anchoring
attracting aare
bacterial
celltomembrane
[61].
cin, like
with other
leaderlessand
bacteriocins,
thought
play a key
roleFurther
in the bacter
investigation into bawcin would be required to determine its specific mode of action.

ocin’s mode of action, particularly in anchoring and attracting a bacterial cell membran

Int. J. Mol. Sci. 2023, 24, 16965

8 of 12

3. Materials and Methods
3.1. Bawcin and Bacterial Strains
Bawcin was chemically synthesized and HPLC-purified by Biomatik, consistent with
its predicted amino acid sequence (MLSFAKLVARLSASKAKWAWNNKGKVVEWIKNGATFEWISNKIDQMMG) and as confirmed by mass spectrometry. B. cereus (Ward’s 85W
0200), B. subtilis (Ward’s 85W 1650), E. faecalis (Ward’s 85W 1100), L. casei (Ward’s 85W
1682), L. lactis ssp. lactis (Ward’s 85W 1774), L. monocytogenes ATCC 19115, M. luteus (Ward’s
85W 0966), S. aureus ATCC 6538, S. epidermidis (Ward’s 85W 1747), S. pyogenes (Ward’s 85W
1180), L. delbruekii ssp. bulgaricus (VWR 470179-126), G. stearothermophilus 1010, P. aeruginosa
(Ward’s 85W 1173), and S. enterica (Ward’s 85W 1710) were used in this study.
3.2. Antimicrobial Activity Testing
The MIC of bawcin against each bacterial strain was determined using the spot-onlawn assay as described previously with modifications [62]. A Mueller Hinton (MH)
double-agar plate was prepared by pouring 5 mL of 0.75% MH agar inoculated with 50 µL
of 0.10 OD625 bacterial suspension onto 20 mL of dried 1.5% MH agar. Bawcin diluted with
sterile water to 128, 64, 32, 16, 8, 4, 2, and 1 µM was spotted in 10 µL aliquots onto the top
agar layer, along with 10 µL of an antibiotic positive control (ampicillin or kanamycin) and
sterile water negative control. Plates were incubated at 37 ◦ C for 16–20 h and the MIC was
identified as the lowest concentration in which a zone of inhibition was observed, where
partial bacterial growth within the zone of inhibition was considered HR. Assays were
performed in triplicate.
3.3. pH Stability Testing
pH screening methods were performed as described previously with modifications [62,63].
The following 100 mM buffer solutions were prepared: glycine-HCl (pH 2), sodium acetate
(pH 4), sodium phosphate (pH 6), Tris-HCl (pH 8), and glycine NaOH (pH 10). Bacteriocinbuffer solutions at various pH values and concentrations were prepared by combining equal
volumes of buffer and peptide solutions. The final concentrations of buffer solutions and
bawcin were 50 mM and half the original bawcin concentration, respectively. To examine
pH stability, bacteriocin-buffer solutions were spotted according to the spot-on-lawn assay
described above following 1 h of incubation at room temperature. M. luteus was used as
the indicator strain, with ampicillin, sterile water, and buffer spotted as positive, negative,
and blank controls, respectively. Assays were performed in triplicate.
3.4. Temperature Stability Testing
The thermal stability of bawcin was examined by incubating aliquots of desired bawcin
concentrations at 25, 37, 60, and 100 ◦ C for 1 h. Residual activity was then measured using
the spot-on-lawn assay method with M. luteus as the indicator strain as described above
with ampicillin and sterile water spotted as positive and negative controls, respectively.
Assays were performed in triplicate.
3.5. Protease Sensitivity Testing
Protease stability testing was evaluated as described previously with modification [64].
Papain (Scholar Chemistry, Rochester, NY, USA), pepsin (Northwest Scientific, Billings, MT,
USA), trypsin (Sigma-Aldrich, Burlington, MA, USA), alpha-chymotrypsin (Worthington,
Lakewood, NJ, USA), and proteinase K (Fisher Scientific, Ottawa, ON, Canada) were
prepared to a final concentration of 1 mg/mL in 50 mM phosphate buffer adjusted to
pH 2 (pepsin), 6 (papain), and 8 (proteinase K, trypsin, alpha-chymotrypsin). Bawcin was
combined with each protease to final concentrations of 1×, 2×, and 4× the MIC. Bawcin’s
residual antimicrobial activity against M. luteus was determined via spot-on-lawn assay
as described above. The following samples were spotted for each assay: (1) protease
combined with bawcin at each concentration, bawcin alone at each concentration, buffer
alone, protease in buffer alone, ampicillin (positive control), and sterile water (negative

Int. J. Mol. Sci. 2023, 24, 16965

9 of 12

control), where all solutions were incubated for 1 h at 37 ◦ C prior to spotting except
ampicillin and sterile water. Assays were performed in triplicate.
3.6. Sequence and Structural Analysis
The three-dimensional structure of bawcin was predicted using SWISS-MODEL [57]
and visualized using PyMOL [58]. A hydrophobic surface map was created using PyMOL and an electrostatic potential map was generated using the APBS extension with the
PDB2PQR molecule preparation method. Primary sequence alignments were performed
using Clustal Omega [18], and sequence parameters were calculated using ExPASy ProtParam [65]. The number of putative transmembrane domains was deduced using the
SOSUI program [19].
4. Conclusions
The present study identified, characterized, and evaluated the novel bacteriocin,
bawcin, the first bacteriocin to be characterized from a B. wiedmannii species. Bawcin
was shown to be a broad-spectrum bacteriocin with considerable stability against pH
and temperature changes, as well as the proteolytic activity of papain and pepsin. Threedimensional structure homology modeling suggests that bawcin adopts a saposin-like fold
with a highly cationic surface that could facilitate its binding to target cell membranes,
leading to subsequent cell death. Given bawcin’s activity and remarkable stability, it has
evident potential for diverse industrial applications, including within the food industry, as
an alternative or additive to existing antimicrobials. Future studies can be directed toward
identifying suitable industrial contexts and optimal conditions for bawcin’s application, as
well as elucidating bawcin’s mode of action.
Author Contributions: Conceptualization, Z.B., J.T.B. and J.Z.A.; methodology, Z.B., J.T.B. and J.Z.A.;
validation, Z.B., J.T.B. and J.Z.A.; formal analysis, Z.B., J.T.B. and J.Z.A.; investigation, Z.B., J.T.B., J.L.
and J.Z.A.; resources, J.Z.A.; data curation, Z.B., J.T.B. and J.Z.A.; writing—original draft preparation,
Z.B., J.T.B. and J.Z.A.; writing—review and editing, Z.B., J.T.B., J.L. and J.Z.A.; visualization, Z.B.,
J.T.B. and J.Z.A.; supervision, J.Z.A.; project administration, J.Z.A.; funding acquisition, J.Z.A. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Natural Sciences and Engineering Research Council of
Canada (RGPIN-2021-02607).
Data Availability Statement: The authors confirm all supporting data, code and protocols have been
provided within the article. The accession number for bawcin is WP_098048139.1 and can be accessed
here: https://www.ncbi.nlm.nih.gov/protein/WP_098048139.1; accessed 10 January 2023.
Conflicts of Interest: The authors declare no conflict of interest.

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