FEMS Microbiology Letters, 363, 2016, fnw092

doi: 10.1093/femsle/fnw092
Advance Access Publication Date: 11 April 2016
Research Letter

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R E S E A R C H L E T T E R – Food Microbiology

Characterization of LysPBC4, a novel Bacillus

cereus-specific endolysin of bacteriophage PBC4
Hongjun Na† , Minsuk Kong† and Sangryeol Ryu∗
Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, Research Institute
of Agriculture and Life Sciences, and Center for Food and Bioconvergence, Seoul National University, Seoul
151-921, South Korea
∗
Corresponding author: Department of Food and Animal Biotechnology, Seoul National University, Seoul 151-921, Korea. Tel: +82-2-880-4856;
Fax: +82-2-873-5095; E-mail: sangryu@snu.ac.kr
†
These authors contributed equally to this work.
One sentence summary: Endolysin from B. cereus-specific phage PBC4 with a narrow host range showed specific lytic activity against the B. cereus group
species without killing B. subtilis or Listeria.
Editor: Andrew Millard

ABSTRACT
Bacillus cereus is a spore-forming, Gram-positive bacterium and is a major food-borne pathogen. A B. cereus-specific
bacteriophage PBC4 was isolated from the soil of a stock farm, and its genome was analyzed. PBC4 belongs to the
Siphoviridae family and has a genome consisting of 80 647-bp-long double-stranded DNA, including 123 genes and two
tRNAs. LysPBC4, the endolysin of PBC4, has an enzymatically active domain (EAD) on its N-terminal region and a putative
cell wall-binding domain (CBD) on its C-terminal region, respectively. Although the phage PBC4 showed a very limited host
range, LysPBC4 could lyse all of the B. cereus strains tested. However, LysPBC4 did not kill other bacteria such as B. subtilis or
Listeria, indicating that the endolysin has specific lytic activity against the B. cereus group species. Furthermore,
LysPBC4 CBD fused with enhanced green fluorescent protein (EGFP) could decorate limited strains of B. cereus group,
suggesting that the LysPBC4 CBD may be a promising material for specific detection of B. cereus.

Keywords: bacteriophage; endolysin; Bacillus cereus; foodborne disease

INTRODUCTION including vomiting and diarrhea (Kotiranta, Lounatmaa and

Haapasalo 2000).
Bacillus cereus is a spore-forming, opportunistic Gram-positive
Bacteriophages have been re-introduced as a biocontrol
bacterium that is widely distributed in the environment. It
agent in the war against B. cereus, particularly with the emer-
is classified as part of the Bacillus cereus group, comprising
gence of multidrug-resistant B. cereus strains (Bottone 2010;
B. thuringiensis, B. anthracis, B. mycoides, B. weihenstaphanensis,
Simm et al. 2012; Lee et al. 2013). These tools can come not only
B. pseudomycoides and B. cytotoxicus, based on their genetic simi-
from a phage itself, but also from phage-encoded proteins, such
larity (Zwick et al. 2012; Guinebretiere et al. 2013). Bacillus cereus
as an endolysin. An endolysin is a peptidoglycan hydrolase of
grows well after cooking and inadequate cooling, since the heat
a phage that lyses its bacterial host at the end of the life cycle
treatment induces spore germination while inhibiting other
(Fischetti 2010). Generally, phages infecting B. cereus show
competing flora (Granum and Lund 1997). The emetic toxins
high specificity toward B. cereus group bacteria, whereas their
and enterotoxins produced by B. cereus cause food poisoning,

Received: 16 February 2016; Accepted: 7 April 2016


C FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

1
 2 FEMS Microbiology Letters, 2016, Vol. 363, No. 12

Table 1. Lytic activity of phage PBC4 and its endolysin LysPBC4, and Bacteriophage isolation and propagation
the binding activity of LysPBC4 CBD.
PBC4 was isolated from soil samples obtained from the National
Species PBC4 LysPBC4 PBC4 CBD Academy of Agricultural Science in Suwon, South Korea. To
isolate a bacteriophage, we applied the same method as in the
B. cereus ATCC 27348 – + +
previous study (Kong and Ryu 2015). Isolated phages were ampli-
B. cereus ATCC 21768 – + –

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fied by serial propagation and concentrated by polyethylene gly-
B. cereus ATCC 13061 – + –
col precipitation and subsequent CsCl density gradient ultracen-
B. cereus ATCC 14579 + + +
trifugation (78 500 × g at 4◦ C for 2 h). The concentrated phages
B. cereus ATCC 21772 – + –
were dialyzed using 2 L of standard dialysis buffer (10 mM NaCl,
B. cereus ATCC 10876 – + +
B. cereus KCTC 3674 – + –
10 mM MgSO4 and 1 M Tris-Cl [pH 8.0]) for 2 h. The phage stock
B. cereus KCTC 13153 – + – obtained was stored in glass vials at 4◦ C.
B. cereus KCTC 1094 – + +
B. thuringiensis ATCC 10792 – + + Host range analysis
B. mycoides ATCC 6452 – + +
B. subtilis ATCC 23857 – – –
Purified phage stock was serially diluted 10-fold using SM buffer
B. subtilis ATCC 6501 – – – (50 mM Tris-Cl [pH 7.5], 0.1 M NaCl, 8 mM MgSO4, 7 H2 O), and
B. licheniformis JCM 2505 – – – 10 μL of each aliquot were spotted onto 0.4% molten top
B. circulans JCM 2504 – – – agar plates containing different strains of bacterial cells. After
B. sphaericus JCM 2502 – – – overnight incubation at 37◦ C, the formation of single plaque on
B. pumilus JCM 2508 – – – the bacterial lawn was recorded.
B. megaterium JCM 2506 – – –
L. monocytogenes ATCC 15313 – – – Transmission electron microscopy analysis
S. aureus RN 4220 – – –
∗
E. coli MG1655 – – – To observe the morphology of PBC4, the phage PBC4 was ob-
∗ served by transmission electron microscopy (TEM). The phage
C. sakazakii ATCC 29544 – – –
PBC4 (∼107 PFU) was dropped onto a carbon-coated copper grid.
+ : positive activity. To negatively stain the virions, PBC4 on the copper grid was in-
– : negative activity. cubated with 2% uranyl acetate 20 s and washed twice with dis-
∗
The Gram-negative bacteria were treated with EDTA (100 mM) for 5 min before
tilled water. The LEO 912AB TEM (Carl Zeiss, Jena, Germany) was
the turbidity assay and CBD-binding assay.
operated at 80 kV, and images of PBC4 were taken by a Proscan
1024 × 1024-pixel charge-coupled device camera with the coop-
endolysins display a broader host range over species or genus eration of the National Academy of Agricultural Science (Suwon,
borders (Loessner et al. 1997; Low et al. 2005; Low et al. 2011; South Korea).
Son et al. 2012; Kong and Ryu 2015). For example, several en-
dolysins of B. cereus phages even showed very high lytic activ- Whole-genome sequencing and genomic analysis
ity against Bacillus species other than the B. cereus group strains
(Low et al. 2005; Yuan, Peng and Gao 2012). However, many Bacil- To extract genomic DNA from PBC4, host DNA was removed by
lus strains (e.g. B. subtilis, B. licheniformis and B. coagulans) clas- treatment of the virions with DNaseI and RNaseA (1 μg mL−1
sified as ‘generally recognized as safe’ (GRAS) are broadly used each) at room temperature for 30 min. The virions were then
as probiotics or in industrial production of enzymes, chemicals lysed by reacting with proteinase K solution [50 μg mL−1 pro-
and food ingredients (Sietske and Diderichsen 1991; Schallmey, teinase K, 20 mM ethylenediaminetetraacetic acid (EDTA), 0.5%
Singh and Ward 2004). Thus, there is a growing need for the sodium dodecyl sulfate (SDS)] at 56◦ C for 1 h. After lysis, the DNA
development of antimicrobial agents against B. cereus group was purified by the phenol-chloroform extraction (Kirby 1956)
strains. and concentrated by ethanol precipitation (Zeugin and Hartley
Here, we analyzed the genomic features of a newly isolated 1985). The purified genomic DNA of the phage PBC4 was se-
B. cereus phage, PBC4, and characterized its endolysin, LysPBC4, quenced using the GS-FLX Titanium sequencer (Roche Holding
which shows highly specific lytic activity against B. cereus group AG, Basel, Switzerland). Total 27 245 sequencing reads obtained
bacteria only. were assembled using the GS De Novo Assembler version 2.6
(Roche Holding AG, Basel, Switzerland) with default parameters
(Table S1, Supporting Information). The position of open read-
MATERIALS AND METHODS ing frames (ORFs) and tRNAs on the genome was predicted by
bioinformatics tools, including Glimmer 3.02 (Delcher et al. 2007),
Bacterial strains and growth conditions
GeneMark (Besemer, Lomsadze and Borodovsky 2001), FGENESB
Bacillus cereus, ATCC 14579, was used as the host bac- (Softberry, Inc., Mount Kisco, NY) and ARAGORN (Laslett and
terium for the isolation and propagation of the bacterio- Canback 2004) software. The function of each ORF was pre-
phage PBC4. Escherichia coli, DH5α and BL21(DE3), were used dicted using NCBI BLASTP and InterProScan (Altschul et al. 1990)
in the cloning and expression of recombinant proteins, re- databases. Based on the informations, each ORF’s name was
spectively. For determination of the antimicrobial spectrum annotated manually. All genes were categorized according to
of phage PBC4 and endolysin LysPBC4, and the binding spec- their function: packaging, structure, host lysis, DNA manipula-
trum of LysPBC4 CBD, 22 bacterial species, including nine tion, additional functions or as hypothetical genes. The genome
B. cereus strains, were used (Table 1). Brain heart infusion map was generated by Genescene software (DNAstar, Madison,
medium (Difco, Detroit, MI) was used for the cultivation of Gram- WI). The gene encoding endolysin was identified, and its do-
positive bacteria, while lysogeny broth medium (Difco) was used main structure was investigated using InterProScan databases
for Gram-negative bacteria. All strains were incubated at 37◦ C. (Altschul et al. 1990).
 Na et al. 3

Phylogenetic analysis Expression and purification of LysPBC4 and its

Phylogenetic trees were drawn to determine PBC4’s phyloge-
netic position among various B. cereus group-specific phages.
The trees were constructed by comparing the amino acid se-
quences of the terminase large subunit (TerL) and the ma-
EGFP-CBD fusion protein
Polymerase chain reaction (PCR) analysis was used to amplify
the LysPBC4 gene and gene fragments encoding LysPBC4 CBD.
The gene encoding LysPBC4 was digested with NcoI-HindIII

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jor capsid protein (MCP) from various phages, respectively.
The Clustal X2 program was used to align all of the se-
quences (Larkin et al. 2007). The MEGA5 software was used
to draw the phylogenetic trees. The trees were created by
the neighbor-joining method and bootstrap analysis (1000
replicates) with P distance values (Kumar, Nei and Dudley
2008).
and inserted into the pET-28a vector (Novagen, Madison, WI).
The gene fragments encoding LysPBC4 CBD were treated with
BamHI-HindIII and subcloned into the pET-28a::EGFP (Kong et al.
2015) to create EGFP-CBD fusion proteins. We constructed two
different EGFP-CBDs, one with a putative linker region and one
without (Fig. 3a). For the purification of each protein, a hexahis-
tidine tag was attached to the C-terminal region of LysPBC4 and

Figure 1. Genomic analysis of phage PBC4. (a) The genome map of bacteriophage PBC4. At least two genes, endolysin and holin, involved in host lysis were confirmed.
Each color of ORF indicates its function: yellow, packaging; blue, structural genes; green, host lysis; red, DNA manipulation; purple, additional functions; and black,
hypothetical genes. The inner circle with the red line indicates the G + C content. The scale unit is a base pair. (b) Comparative genomic analysis between phage PBC4
and Basilisk on the nucleotide sequence level. Both phages are B. cereus-targeting Siphoviridae. Their genomes have high similarities at the nucleotide or protein level.
However, some of the genes, like those for the tail fiber, differ, indicating that these two phages may have different host ranges or lytic activities.
 4 FEMS Microbiology Letters, 2016, Vol. 363, No. 12
the N-terminal region of the EGFP-CBD fusion proteins, respec-
tively. The expression of proteins was induced by treating the
E. coli BL21(DE3) strains containing recombinant plasmids with
1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-
Aldrich, St. Louis, MO). The LysPBC4 and EGFP CBD fusion pro-
teins were expressed by incubating the cells at 37◦ C for 6 h and at
18◦ C for 20 h, respectively. After centrifugation (5000 × g at 4◦ C
for 5 min), the supernatant was discarded and the remaining
pellet was resuspended with 5 mL of lysis buffer (50 mM Tris-
Cl [pH 8.0], 200 mM NaCl). Cells were lysed by sonication (Bio-
Rad Laboratories, Inc., Hercules, CA) for 20 min, with intervals of
15 s on/off. The insoluble material was discarded by centrifuga-
tion (21 000 × g at 4◦ C for 20 min), and the supernatant was col-
lected, mixed with 500 μL of Ni-NTA resin and incubated at 4◦ C
for 1 h with gentle shaking. The flow-through was discarded and
the resin was serially washed with 10 mL of 10 mM imidazole
and 5 mL of 20 mM imidazole. An elution buffer (50 mM Tris-
Cl [pH 8.0], 200 mM NaCl and 210 mM of imidazole) was used
to elute the protein. The concentration of proteins was deter-
mined by Bradford assay, and the purity was confirmed by SDS-
polyacrylamide gel electrophoresis (PAGE) (Dunne et al. 2010).
Purified protein was stored in storage buffer (50 mM Tris-Cl [pH
8.0], 200 mM NaCl, 50% glycerol) at −20◦ C.
Turbidity reduction assay
Turbidity reduction assay was performed using the same
method as in the previous study (Kong and Ryu 2015). Each assay
was conducted in triplicate.
CBD-binding assay
Binding activities of all EGFP-CBD, fusion proteins were tested as
described earlier (Loessner et al. 2002). DE/Axio Imager A1 epi-
Figure 2. Phylogenetic tree of various phages having (a) related major capsid proteins (MCPs) and (b) TerLs. Each host bacterium is shown in the parentheses after the
name of the phage. The numbers at the nodes represent the bootstrap probabilities.
fluorescent microscopy (Carl Zeiss, Oberkochen, Germany) was
used in the assay.
GenBank accession numbers
The GenBank accession numbers of PBC4 and LysPBC4 are
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KT070866 and AKQ08217, respectively.
RESULTS AND DISCUSSION
Morphological and antibacterial properties of
phage PBC4
The B. cereus phage PBC4 was isolated from soils. TEM analysis
revealed that PBC4 was classified into the Siphoviridae, consisting
of a capsid with a diameter of 65 ± 10 nm and a non-contractile
tail with a length of 430 ± 30 nm (Fig. S1, Supporting Informa-
tion). PBC4 showed high host specificity, producing plaques only
against a single strain of B. cereus out of the nine B. cereus strains
tested (Table 1). Other Gram-positive or Gram-negative bacteria
were resistant to PBC4.
Analysis of complete genome sequence of phage PBC4
The genome of phage PBC4 comprises double-stranded DNA,
consisting of 80 647 bp of nucleotides with G + C contents of
34%. Bioinformatic analysis revealed that the PBC4 genome has
123 ORFs and two tRNAs. The predicted ORFs were classified
into six different groups according to their function: phage DNA
packaging, virion structure, host lysis, DNA manipulation, ad-
ditional functions and hypothetical proteins (Fig. 1a). The exis-
tence of an integrase, containing a DNA breaking-rejoining do-
main (PBC4 072) with homology to the tyrosine recombinase
XerD of phage Basilisk (93% amino acid sequence similarity;
GenBank accession no. KC595511.2) (Grose et al. 2014b) and the
 Na et al. 5

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Figure 3. Modular structure of LysPBC4. (a) Schematic representation of LysPBC4 and EGFP CBD fusion proteins. LysPBC4 has an EAD on its N-terminus and a putative
CBD on its C-terminus. The linker region was predicted by bioinformatic analysis. The putative linker sequence was included in EGFP CBD1, while it was absent in
the EGFP CBD2. Each number corresponds to the positions of amino acid residues. (b) Sequence alignment of LysPBC4-related endolysins. Conserved and identical
residues are shaded in gray (dark gray, >70% conserved; light gray, >40% conserved) and black, respectively. Red arrowheads indicate that the position of conserved
active sites and blue arrowheads represent the zinc ion-binding sites. (c) Sequence alignment between endolysins of PBC1 and PBC4. Conserved residues are shaded
in black.

integrase of Bacillus phage Staley (59% amino acid sequence sim-
ilarity; GenBank accession No. NC 022767) (Hastings et al. 2013),
suggests that PBC4 is a temperate phage having both lytic and
lysogenic life cycles. Although no MCP was identified by se-
quence analysis, the protein PBC4 006 could be predicted to be
an MCP, because it showed high sequence identity (71%) with
that (gp36) of the B. cereus phage Basilisk, whose MCP was exper-
imentally proven (Grose et al. 2014a,b). A putative TerL belong-
ing to the terminase 1 superfamily was identified, but a small
subunit of terminase-encoding gene was not found. Among the
predicted phage’s structural proteins, a putative tail fiber protein
(PBC4 021) corresponds to the short tail fiber observed in a TEM
image (Fig. 1 and Fig. S1, Supporting Information).
The comparative analysis of genomes between PBC4 and
Basilisk revealed that they share an 83.3% nucleotide identity
over the entire genome with the same architectures (Fig. 1b).
This is also evident from phylogenetic trees based on MCP and
TerL, which are common targets for phage taxonomy (Brüssow
and Frank 2001; Comeau and Henry 2008) (Fig. 2). Both MCP and
TerL of PBC4 have high similarities to those of Basilisk, with 71%
and 82% amino acid sequence identity, respectively. These data
indicate that the phage PBC4 and Basilisk are closely related
phages. Interestingly, although the late gene products, such as
the terminase, structural proteins and lysis-related proteins of
PBC4 and Basilisk, demonstrated high sequence homology, their
putative tail fiber proteins showed relatively low sequence iden-
tity (41%). Specifically, a putative chitinase, which was found ad-
jacent to the tail fiber protein of Basilisk, is absent in PBC4. Con-
sidering that the tail fiber proteins of phages have been known
to determine the host range of phages (Scholl et al. 2001), the
host specificities of the two B. cereus phages might differ.
Identification of endolysin from PBC4 and its domain
composition
We identified an N-acetylmuramoyl-L-alanine amidase
(PBC4 025), which has lytic activity against peptidoglycan
cell walls (Young 1992; Young, Wang and Roof 2000), and anno-
tated it as an endolysin. The endolysin of phage PBC4 (LysPBC4)
has two distinct domains. On the N-terminal region, the domain
‘amidase 3’ (PF01520), generally functioning as an enzymat-
ically active domain (EAD) (Ghuysen et al. 1969; Herbold and
Glaser 1975), was predicted, while the “amidase02 C” domain
(PF12123), a putative cell wall-binding domain (CBD) (Yuan, Peng
and Gao 2012; Kong et al. 2015), was found from the C-terminal
region (Fig. 3a). These two distinct domains appeared to be
connected by a flexible linker region, since the region from the
170th to 185th amino acid is rich in glycine, alanine having
short side chains, serine, threonine and asparagine having polar
side chains, all of which are predicted to form random coils and
are usually found in many linker proteins (Schmitz, Schuch and
 6 FEMS Microbiology Letters, 2016, Vol. 363, No. 12
Figure 4. Lytic activities of LysPBC4. (a) To optimize the amount of endolysin in the turbidity reduction assay, different amounts of LysPBC4 were treated to the
exponentially growing B. cereus ATCC 14579. Filled diamond, 15 μg; filled triangle, 5 μg; filled square, 1 μg; filled circle, control group. We used 15 μg of LysPBC4 because
it was the minimum amount to effectively kill B. cereus ATCC 14579. (b–d) Equal amounts of LysPBC4 (15 μg) were added to the cultures of (b) B. cereus ATCC 27348, (c) B.
subtilis ATCC 23857 and (d) Listeria monocytogenes ATCC 15313; filled circle, LysPBC4-treated bacterial cell culture; filled square, control group. All experiments were
performed in triplicate. The error bars indicate the standard error of the mean.
Fischetti 2010; Schmelcher, Tchang and Loessner 2011). The
active site residues His10, Glu24, His80 and Glu142 were found
to be conserved in EAD; three of these residues (His10, Glu24
and His80) were predicted to be zinc-binding sites.
BLASTP analysis revealed many proteins having sequence
homology with LysPBC4. Most were autolysins, cell wall-
hydrolyzing enzymes (Shockman and Höltje 1994) required for
bacterial cell growth, peptidoglycan maturation, and various
other cellular functions (Blackman, Smith and Foster 1998;
Smith, Blackman and Foster 2000). Additional comparative anal-
ysis revealed that the N-terminal EAD of LysPBC4 is more con-
served than the C-terminal CBD. For instance, the N-terminal
regions of two endolysins from phage PBC4 and Basilisk showed
69% sequence identity, whereas the C-terminal regions showed
only 33% sequence homology. Due to the conserved EAD,
LysPBC4 showed an overall 63% amino acid sequence identity
with an endolysin of Basilisk (Grose et al. 2014a,b), 51% with an
endolysin of BCP78 (GenBank accession no. JN797797) and 49%
with PlyM19 (GenBank accession no. HM011607.1) (Schmitz et al.
2010) (Fig. 3b). LysPBC4 also showed close relatedness (30% iden-
tity) to LysPBC1, as they have similar domain structures (ami-
dase 3 and amidase02 C) (Kong et al. 2015). However, the CBDs of
these endolysins showed only marginal sequence identity (23%)
(Fig. 3c). This result implies that some key residues or motifs in
both CBDs are conserved (which is why they are predicted as
same amidase02 C domain), whereas their overall structures or
binding targets may differ.
Antibacterial activity of LysPBC4
The LysPBC4 was expressed and purified from a recombinant
E. coli BL21(DE3) strain (Fig. S2, Supporting Information). A tur-
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bidity reduction assay revealed that the endolysin LysPBC4
showed broader lytic activity than the host range of bacterio-
phage PBC4, but it was still specific for B. cereus group strains (Ta-
ble 1). LysPBC4 could kill all of the B. cereus strains tested, and it
could also lyse the B. thuringiensis and B. mycoides strains, which
belong to the B. cereus group. Meanwhile, other Bacilli, including
B. subtilis, were resistant to LysPBC4. Other Gram-positive and
Gram-negative bacteria were also insensitive to LysPBC4, mean-
ing that the endolysin specifically kills the B. cereus group at
the species level (Table 1, Fig. 4). Considering that some Bacillus
species, such as B. subtilis, have been granted GRAS status (West-
ers, Westers and Quax 2004) and are widely used as the starter
strain for fermented foods (Cladera-Olivera, Caron and Brandelli
2004), the B. cereus-specific lytic activity could render LysPBC4 a
promising tool for controlling pathogenic Bacilli.
Binding spectrum of LysPBC4 CBD
To confirm the binding activity of the C-terminal amidase 02
domain, two different length variants (one with a puta-
tive linker and one without) were tested (Fig. 5). Both con-
structs were tagged with EGFP at the N-terminus and named
EGFP PBC4 CBD1 and EGFP PBC4 CBD2, respectively. As shown
in Fig. 5, the EGFP PBC4 CBD1, which includes a putative linker
sequence, displayed a strong cell wall-binding activity, whereas
the EGFP PBC4 CBD2, which devoid of the putative linker,
showed no binding activity. These results indicate the impor-
tance of a putative linker region for cell wall-binding capacity
of EGFP-tagged LysPBC4 CBD. It could be that the putative linker
region is critical for proper folding of the LysPBC4 CBD. It is also
possible that the linker itself plays an important role in bind-
ing cell wall of the B. cereus. Further, study would be necessary

Figure 5. Binding activity of LysPBC4 CBD. (a–b) Images of cells of B. cereus ATCC 27348 treated with EGFP CBD1 fusion protein. (a) Light microscopic image and
(b) epifluorescent image were taken at the same position. (c–d) Images of same bacterial cells treated with EGFP CBD2 fusion protein, EGFP-fused CBD without linker.
(c) Light microscopic image and (d) epifluorescent image of EGFP CBD2-treated B. cereus. ATCC 27348 were taken at the same position. The scale bar is presented at
lower edge of each figure.
to elucidate the exact role of the linker region of LysPBC4. Bind-
ing spectrum of EGFP PBC4 CBD showed that LysPBC4 CBD could
bind to four of nine strains of B. cereus and two other B. cereus
group species tested (Table 1). Other bacteria, such as B. subtilis
and other bacterial species, could not be bound by LysPBC4 CBD
(Fig. S3, Supporting Information). These results suggest that
B. cereus group-specific cell wall moieties are the most likely
binding target of LysPBC4 CBD. Also, the inability of binding of
LysPBC4 CBD toward certain B. cereus strains proposes the differ-
ence of cell wall structure among the B. cereus group strains. The
specific binding activity of LysPBC4 CBD could be useful for the
development of efficient bioprobes against B. cereus.
SUPPLEMENTARY DATA
Supplementary data are available at FEMSLE online.
FUNDING
This work was supported by the National Research Foundation
of Korea (NRF) grant (NRF-2014R1A2A1A10051563) and the Pub-
lic Welfare & Safety research program (NRF-2012M3A2A1051684)
funded by the Ministry of Science, ICT and Future Planning,
Republic of Korea.
Conflict of interest. None declared.
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