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Applied and Environmental Microbiology, December 2000, p. 5480-5483, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cloning, Nucleotide Sequence, and Expression of the
Gene Encoding the Bacteriocin Boticin B from Clostridium
botulinum Strain 213B
Sean S.
Dineen,
Marite
Bradshaw, and
Eric A.
Johnson*
Department of Food Microbiology and
Toxicology, Food Research Institute, University of Wisconsin,
Madison, Wisconsin 53706
Received 26 June 2000/Accepted 19 September 2000
 |
ABSTRACT |
Boticin B is a heat-stable bacteriocin produced by
Clostridium botulinum strain 213B that has inhibitory
activity against various strains of C. botulinum and
related clostridia. The gene encoding the bacteriocin was localized to
a 3.0-kb HindIII fragment of an 18.8-kb plasmid, cloned,
and sequenced. DNA sequencing revealed the boticin B structural gene,
btcB, to be an open reading frame encoding 50 amino acids.
A C. botulinum strain 62A transconjugant containing the
HindIII fragment inserted into a clostridial shuttle vector
expressed boticin B, although at much lower levels than those observed
in C. botulinum 213B. To our knowledge, this is the first
demonstration and characterization of a bacteriocin from toxigenic
group I C. botulinum.
| |
TEXT |
Bacteriocins are antimicrobial
peptides or proteins formed by certain bacterial species that typically
have inhibitory activity against closely related organisms
(5). C. botulinum is a gram-positive, endospore-forming anaerobic bacterium that causes the severe
neuroparalytic illness known as botulism in humans and animals
(10). Our laboratory demonstrated that Clostridium
botulinum strain 213B produces a heat-stable bacteriocin, called
boticin B, that is bactericidal to certain C. botulinum
strains and related species (13; H. Sugiyama, E. A. Johnson, R. Sandler, and S. Cole, unpublished data). Purification and
characterization of the boticin B peptide was performed in this
laboratory (13) and will be described in a separate communication.
Isolation of the boticin B structural gene.
In order to
determine the location of the boticin B gene, plasmid DNA and total
genomic DNA (containing plasmid and chromosomal DNAs) were isolated
from C. botulinum strain 213B. C. botulinum 213B
contains two plasmids (18.8 and 12.1 kb). Undigested plasmid DNA and
HindIII- and EcoRI-digested plasmid and total
DNAs were separated on an agarose gel by electrophoresis, transferred
to a nylon membrane, and hybridized to a degenerate 18-mer
oligonucleotide (BB1) (5'-GC[A/
T]GT[A/T]AATGAATTTGT[A/T]-3') probe. The sequence of the
probe was designed based on the previously identified amino acid
sequence of boticin B chymotryptic fragments (13) and the preferential codon usage in clostridia (9, 12). The selected region contained the fewest number of degenerate nucleotides. The probe
hybridized to the 18.8-kb plasmid of C. botulinum 213B (Fig.
1, lanes A), a 3.0-kb
HindIII plasmid DNA fragment (lanes C), and a 9.0-kb
EcoRI plasmid DNA fragment (lanes B). These hybridization signals were also the only signals observed in HindIII-
or EcoRI-digested total DNA (Fig. 1, lanes E and D),
indicating that the boticin B gene is located on the 18.8-kb plasmid.
Thus, similar to many bacteriocin genes of gram-positive bacteria
(5), the boticin B gene is located on a plasmid.
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FIG. 1.
Isolation of the boticin B structural gene,
btcB. Agarose gel electrophoresis (left panel) and Southern
blot hybridization (right panel) were performed using undigested
plasmid DNA and plasmid and total genomic DNAs isolated from C. botulinum strain 213B digested with EcoRI or
HindIII. Lanes: A, undigested plasmid DNA; B, plasmid
DNA digested with EcoRI; C, plasmid DNA digested with
HindIII; D, total genomic DNA digested with
EcoRI; E, total genomic DNA digested with
HindIII. Lanes M1 ( DNA digested with
HindIII) and M2 (1-kb DNA ladder) represent molecular
weight markers, with sizes in kilobases.
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|
Structure of the btcB gene.
The 3.0-kb
HindIII fragment containing the boticin B gene was
subcloned into pBluescript KS II(+), yielding pMVP924. The location of
the boticin B gene within the fragment was determined by detailed restriction mapping and hybridization with the BB1 probe. The DNA
sequence of the first 1,000 bp of the HindIII fragment
from the C. botulinum 213B 18.8-kb plasmid containing the
btcB gene is shown in Fig. 2.
The length of the btcB structural gene is 150 bp, and it
encodes a 50-amino-acid protein with a calculated molecular mass of
5,138 Da. A putative ribosome binding site (RBS), AAGGAGG, is located 8 bp upstream of the start codon of the btcB gene. Sequences
exhibiting similarity to a promoter region are located upstream of the
RBS (nucleotides 503 to 550) (Fig. 2). A region of dyad symmetry that
could form a stem-loop structure is located downstream of the
btcB gene (nucleotides 730 to 745 and 753 to 768) (Fig. 2).
This region has the features associated with bidirectional
rho-independent transcription termination (8). Sequence
analyses revealed several open reading frames (ORFs) in the vicinity of
the btcB gene. One of these, ORF-30, is located just 63 bp
downstream of the btcB gene (Fig. 2). Interestingly, the
30-amino-acid putative peptide encoded by this ORF contains 20%
lysine, 17% phenylalanine, 10% serine, and 10% glutamic acid residues. However, it is not known if ORF-30 is transcribed as a
monocistronic message or is part of an operon with btcB and possibly other bacteriocin-associated genes such as genes coding for
regulation, immunity, extracellular translocation, and amino acid
modification, as has been described for certain other bacteriocin genetic systems (5, 6). No protein in the GenBank databases revealed significant homology to boticin B, the ORF-30 product, or
products of other ORFs surrounding btcB.
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FIG. 2.
Nucleotide sequence of the first 1,000 bp of the 3.0-kb
HindIII fragment from the C. botulinum 213B
18.8-kb plasmid. The deduced amino acid sequences of the
btcB gene and ORF-30 are shown below the nucleotide
sequence. The location of oligonucleotide BB1, the NdeI
site, and the putative RBS are shown (marked and underlined).
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The
btcB gene encodes a 50-amino-acid protein with a
calculated molecular mass of 5,138 Da. Amino acid analysis of purified
boticin B estimated that the active protein contained 39 amino
acids,
and mass spectroscopic analysis determined its molecular
mass to be
4,003 Da (
13). The smaller size of the purified protein
could indicate that the
btcB gene encodes a prepeptide that
is
posttranslationally modified to form the active peptide, which
is
relatively common for bacteriocins from other gram-positive
bacteria
(
5). The amino acid sequence of one of the chymotrypsin
fragments (11 amino acids) analyzed previously did not contain
the
first 10 amino acids as deduced from the gene (Fig.
3). Interestingly,
this region does not
contain a chymotrypsin cleavage site, indicating
that these amino acids
may not be part of the active protein.
The molecular mass of the
peptide without these 10 amino acids
would correlate with the molecular
mass of 4,003 Da as determined
previously by mass spectroscopy. In
addition, the peptide started
with a valine residue, not asparagine,
which is in this position
according to the nucleotide sequence of the
btcB gene (Fig.
3).
The other chymotryptic peptide, a
14-amino-acid fragment, is missing
a stretch of three amino acid
residues (Asn-Leu-Asp) that are
encoded by the
btcB gene
(Fig.
3). However, it is not clear whether
these amino acid residues
are deleted from the mature peptide
or whether the discrepancy is due
to technical difficulties involved
in sequencing this very hydrophobic
peptide fragment. These findings
further support the possibility that
the boticin B encoded by
the
btcB gene is synthesized as a
prepeptide, followed by cleavage
of leader peptide sequences, and
further undergoes several modifications
before assuming its active
form.
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FIG. 3.
Alignment of chymotryptic peptide fragment sequences and
the putative boticin B sequence. The sequences of the 11- and
14-amino-acid chymotryptic fragments isolated by Yu (13) are
underlined. The arrows indicate chymotrypsin cleavage sites.
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Expression of the boticin B gene in C. botulinum
strain 62A. Experiments designed to express boticin B in
Escherichia coli were unsuccessful (data not shown);
therefore, we attempted
to express the bacteriocin in a strain of
C. botulinum that does
not produce boticin B. The 3.0-kb
HindIII fragment containing
the
btcB gene
with its putative regulatory sequences was inserted
into the
mobilizable plasmid pJIR1457 (
7), yielding pMVP1113.
The
plasmid pMVP1113 was transferred into
C. botulinum strain
62A by conjugation from the
E. coli donor strain
S17-1(pMVP1113)
as described previously (
2).
The
C. botulinum 62A transconjugant carrying
btcB
expression construct pMVP1113 was analyzed for boticin B production
using
a well diffusion assay. The transconjugant containing vector
pJIR1457
alone was used as a negative control.
C. botulinum
strain 213B
and
C. botulinum transconjugants were grown in
M1 broth (5% Trypticase
peptone, 2.5% proteose peptone, 1% glucose,
2% yeast extract,
and 0.1% sodium thioglycolate) without antibiotics
for 3 days
at 37°C. Bacterial cells were removed by centrifugation,
and the
culture supernatants were filtered through a
0.2-µm-pore-size
membrane filter. Fifty-microliter culture filtrates
of 62A(pMVP1113),
62A(pJIR1457), and
C. botulinum
strain 213B were loaded into precut
wells in M1 agar plates, incubated
anaerobically for 6 h at room
temperature to allow inhibitory
agents to diffuse through the
agar medium, and then overlaid with 6 ml
of M1 broth containing
0.75% agar and ca. 10
6 cells of
C. botulinum strain 62A per ml. The plates were incubated
anaerobically for 24 h at 30°C and examined for the presence of
inhibition zones surrounding the wells. No inhibitory activity
was
detected in the culture media of both transconjugant strains,
while
strain 213B exhibited inhibitory activity (data not shown).
To
determine if bacteriocin was produced but not released into
the culture
media by the
C. botulinum 62A transconjugant carrying
boticin B expression construct pMVP1113, bacterial cells were
sonicated
for 10 min and cell-free lysates were also analyzed
for boticin B
production. However, no inhibitory activity was
observed (data not
shown). To test whether boticin B was expressed
at levels too low to
detect inhibitory activity in culture filtrates,
culture filtrate
proteins were concentrated by ammonium sulfate
precipitation. Ammonium
sulfate was added to 63% saturation to
150 ml of culture filtrates and
incubated for 18 h at 4°C to allow
precipitation of proteins
(
1). Precipitated proteins were collected
by centrifugation
and dissolved in 1 ml of 25 mM sodium phosphate
buffer (pH 6.8), and 50 µl was added to wells in M1 agar plates.
The 150-fold-concentrated
culture filtrate of 62A(pMVP1113) produced
an inhibition zone in well
diffusion plates overlaid with
C. botulinum 62A (Fig.
4). The inhibition zone (2 mm) was
smaller than that
observed with the 213B culture filtrate (8 mm). The
concentrated
culture filtrate of 62A(pJIR1457) did not reveal any
inhibitory
activity, indicating that the activity of 62A(pMVP1113) was
due
to the presence of the 3.0-kb
HindIII fragment from
C. botulinum strain 213B containing the boticin B gene. The
low levels of phenotypic
expression of boticin B that were observed in
the
C. botulinum strain 62A transconjugant could possibly be
explained by the lack
of additional genes needed for expression of
functional boticin
B, such as regulatory genes or genes coding for
immunity, peptide
modification, and extracellular translocation, in
this heterologous
strain.
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FIG. 4.
Expression of the boticin B gene in C. botulinum strain 62A. Culture filtrates were tested for inhibitory
activity in well diffusion plates. A, C. botulinum 213B; B,
C. botulinum 62A(pMVP1113); C, C. botulinum
62A(pJIR1457). The latter two culture filtrates were concentrated
150-fold by ammonium sulfate precipitation.
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The presence of sequences homologous to
btcB was examined in
other clostridial strains. Total genomic DNA was isolated from
the
following strains:
C. botulinum strains 62A, Hall A, 588 Ab,
NCTC 2916 A(B), 2B, 17B, Okra B, Alaska E, Kyoto F, and Langeland
F;
Clostridium baratii strain ATCC 27638;
Clostridium
butyricum strains 1024 and ATCC 19398; and
Clostridium
sporogenes strain
PA3679.
HindIII- and
EcoRI-digested total DNA from each strain
was separated on
an agarose gel by electrophoresis, transferred
to a nylon membrane, and
hybridized to the BB1 oligonucleotide
probe.
C. botulinum
strain 213B
HindIII- and
EcoRI-digested total
DNAs were included as positive controls. The probe hybridized
to the
same-size 3.0-kb
HindIII and 9.0-kb
EcoRI DNA
fragments
from strains 213B and 588 Ab. No hybridization was observed
with
chromosomal or plasmid DNA from the other clostridial strains
(data not shown). Plasmid DNA was isolated from strain 588 Ab
and
compared to plasmids from strain 213B. Both strains contained
two
similar-sized plasmids, and their
HindIII and
EcoRI digestion
band profiles were identical (data not
shown). These data suggest
that strain 588 Ab carries the same plasmids
as strain 213B or
closely related plasmids. Culture filtrates of strain
588 Ab,
similar to those of strain 213B, were inhibitive to growth of
C. botulinum strain 62A (data not shown). Interestingly,
culture
filtrates of strain 213B were also inhibitive to growth of
strain
588 Ab (data not shown), indicating that strain 213B may produce
other bacteriocins. Although
btcB is located on a plasmid,
the
absence of
btcB in the variety of strains analyzed
suggests that
the plasmid is not frequently transferred among the
clostridia.
However, we feel that it would be of interest to examine
other
clostridial strains that cause intestinal infections, such as
strains of
Clostridium difficile and
Clostridium
perfringens,
for the presence of
btcB.
To our knowledge, these data provide the first genetic evidence of a
bacteriocin being produced by a toxigenic strain of proteolytic,
group
I
C. botulinum. Boticin B appears to be unique since it
shows no detectable homology to other bacteriocins, including
those
from clostridial species (
4). The production of boticin
B
could have important practical implications in regard to intestinal
and
infant botulism. Although it has been suspected that
C. botulinum strains that have the capability to colonize the infant
and microbially
altered adult intestinal tracts produce substances with
antibiosis
properties against competitors (
10), inhibitory
compounds other
than fermentation end products have not been previously
reported.
Also, since
C. botulinum 213B is commonly used in
strain mixture
cocktails for challenge studies in foods (
3,
11), it is possible
that the production of boticin B could
inhibit other
C. botulinum strains in the mixture,
contributing to low toxin production in
a permissive food environment.
Based on our results, it may be
useful to evaluate
C. botulinum strains in a food inoculum mixture
for antagonistic
activity.
Nucleotide sequence accession number.
The nucleotide sequence
presented in this paper was submitted to GenBank and was given the
accession number AF278540.
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ACKNOWLEDGMENTS |
This work was financially supported by the National Institutes of
Health (grant AI42226), by the USDA (grants 9802799 and 9635201), by a
Hatch grant (3571), by industry sponsors of the Food Research
Institute, and by the College of Agricultural and Life Sciences,
University of Wisconsin-Madison.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Food Microbiology and Toxicology, Food Research Institute, University of Wisconsin, 1925 Willow Dr., Madison, WI 53706-1187. Phone: (608)
263-7944. Fax: (608) 263-1114. E-mail:
eajohnso{at}facstaff.wisc.edu.
| |
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Applied and Environmental Microbiology, December 2000, p. 5480-5483, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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