Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Characterization, N-terminal sequencing and classification

of cerein MRX1, a novel bacteriocin purified from a newly
isolated bacterium: Bacillus cereus MRX1
S. Sebei1, T. Zendo1, A. Boudabous2, J. Nakayama1 and K. Sonomoto1
1 Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of
Agriculture, Graduate School, Kyushu University, Fukuoka, Japan
2 Laboratoire de Microbiologie, Departement de Biologie, Faculte des Sciences de Tunis, Campus Universitaire, Tunis, Tunisia

Keywords
antimicrobial peptide, Bacillus cereus,
bacteriocin, purification, sequencing.
Correspondence
S. Sebei, Laboratory of Microbial Technology,
Division of Microbial Science and Technology,
Department of Bioscience and Biotechnology,
Faculty of Agriculture, Graduate School,
Kyushu University, 6-10-1 Hakozaki,
Higashi-ku, Fukuoka 812-8581, Japan.
E-mail: sebeisamir@yahoo.fr

2006 1771: received 15 December 2006,

revised 20 February 2007, and accepted
6 March 2007
doi:10.1111/j.1365-2672.2007.03395.x

Abstract
Aim: To purify and characterize the bacteriocin produced by strain MRX1.
Methods and Results: A bacteriocin-producing strain was isolated and identified as Bacillus cereus. The bacteriocin, called cerein MRX1, was purified from
the culture supernatant using hydrophobic interaction, cation-exchange chromatography and RP-HPLC. It could also be purified in abundance from the
cell surfaces of the producer strain. Mass spectrometry revealed its molecular
mass of 313793 Da. Sequencing of chemically modified bacteriocin identified
its partial sequence: DWTCWSCLVCAACSVELL. Amino acid analysis, confirmed by 1H-NMR, suggested cerein MRX1 to be a class II bacteriocin. This
bacteriocin was remarkably hydrophobic, heat-stable and could withstand a
wide range of pH. It exhibited a bactericidal mode of action against Bacillus
coagulans JCM 2257T. Cerein MRX1 was especially active against spoilage bacteria such as Bacillus subtilis and Listeria innocua (MICs in the 1 lg ml)1
range). In contrast, lactic acid bacteria were resistant or required higher concentrations to be inhibited.
Conclusions: Cerein MRX1 is similar by its N-terminal sequence to thuricin 17
recently isolated from Bacillus thuringiensis NEB17. However, the two bacteriocins are different by their molecular masses and amino acid compositions.
Significance and Impact of the Study: Chemical stability of cerein MRX1 and
its ability to inhibit a large number of undesirable bacteria may give an advantage to its food or clinical application as an antibacterial agent.

Introduction
Bacteria produce a variety of antimicrobial substances that
are able to kill or inhibit other micro-organisms. Bacterial
antibiotics can be subdivided into two types on the basis
of their chemical nature: (i) nonpeptide antibiotics such
as the antibacterial and antifungal phospholipid bacilysocin produced by Bacillus subtilis 168 (Tamehiro et al.
2002); and (ii) peptide antibiotics, which fall into two
large groups differentiated on the basis of the biosynthetic
pathways by which they are generated. One group comprises nonribosomally synthesized peptides produced on
large enzymatic complexes. These peptides consist of only

few amino acid residues. For example, surfactin produced

by various strains of B. subtilis is composed of seven
amino acids (Carrillo et al. 2003). The second group comprises ribosomally synthesized peptides (i.e. bacteriocins).
Those produced by Gram-positive bacteria have typically
2060 amino acid residues. Bacteriocins produced by
Gram-positive bacteria exhibit a number of characteristics
that make them attractive for both the food industry and
biomedical applications (Bower et al. 2001; Chen and
Hoover 2003). Determination of the amino acid sequence
of bacteriocins can often be challenging. In fact, the standard Edman sequencing techniques are usually unable
to sequence through unusual amino acid residues such as

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Purification and characterization of cerein MRX1

dehydro residues, and are hindered by intramolecular

linkages such as the thioether ring in sublancin 168 (Paik
et al. 1998) and the disulfide bridge in cerein 7B (Oscariz
et al. 2006). Cotter et al. (2005) distinguished two classes
of bacteriocins: class I comprises lanthionine-containing
peptides (also called the lantibiotics). Mature lantibiotics
typically contain the unusual (i.e. not genetically encoded)
dehydro amino acids dehydroalanine (Dha) and dehydrobutyrine (Dhb). Class II comprises nonlanthionine-containing peptides. They are categorized into four subclasses.
Subclass IIa bacteriocins, such as pediocin AcH (Motlagh
et al. 1992) show narrow spectra and high inhibitory
activity against Listeria monocytogenes. They are structurally characterized by the consensus sequence YGNGV in
their N-terminal region. Subclass IIb includes bacteriocins
whose activities depend on the complementary action of
more than one peptide, the most often two (e.g. lactacin
F, Allison et al. 1994). Subclass IIc consists of circular bacteriocins such as circularin A (Kemperman et al. 2003)
and subclass IId consists of non-pediocin-like single linear bacteriocins (e.g. divergicin A, Worobo et al. 1995). In
the last few years, the number of scientific reports dealing
with Bacillus antimicrobial peptides classifiable as bacteriocins has appreciably increased. The newly characterized
bacteriocins include the lantibiotics ericin A (2986 Da)
and ericin S (3342 Da) produced by B. subtilis (Stein et al.
2002); cerein 7A (3940 Da) and cerein 7B (4893 Da) from
Bacillus cereus (Oscariz et al. 1999; Oscariz et al. 2006)
and the subclass IIa peptide SRCAM 1580 (3486 Da) produced by Bacillus circulans (Svetoch et al. 2005). Interestingly, many bacteriocins produced by Bacillus species were
shown to have unique unusual structures and novel biological activities. For example mersacidin inhibits cell wall
biosynthesis by targeting the murein precursor lipid II at a
molecular site not targeted by all known antibiotics (Brotz
et al. 1998). Subtilosin A was shown to have unique intramolecular linkages between cysteine sulfurs and a-carbons
of Phe and Thr (Kawulka et al. 2004); and sublancin 168
is the only known lantibiotic with disulfide bridges (Paik
et al. 1998). Here, we report about the structural and
biological characterization of cerein MRX1, a bacteriocin
produced by a newly isolated bacterium: B. cereus MRX1.
Materials and methods
Bacterial strains and culture conditions
Bacterial strains and their growth conditions are listed in
Table 1. Culture media were from Becton Dickinson
(Sparks, MD, USA) and Merck (Darmstadt, Germany).
Solid media and soft agar media were prepared by adding
15% and 06% agar to broth media, respectively. Bacteria
were kept as frozen cultures in 25% glycerol at )70C.
1622

S. Sebei et al.

Bacterial identification
Sugar fermentation profiling of strain MRX1 was carried
out by means of API 50 CHB (bioMerieux, Marcy
lEtoile, France). Total DNA was extracted according to
Kalman et al. (1993). Two primers were used for 16S
rDNA analysis: MRXF1: 5-GCGGCGTGCCTAATACATGC-3 and MRXR2: 5-CTCTACGCATTTCACCGCTAC-3. PCR was performed using Ex Taq polymerase
(Takara, Otsu, Japan) under the following conditions:
94C for 3 min followed by 30 cycles of denaturation at
94C for 30 s, annealing at 56C for 30 s and polymerization at 72C for 1 min. Two primers (Manzano et al.
2003) were used for gyrB gene analysis: BCFW1: 5GTTTCTGGTGGTTTACATGG-3 and BCRW1: 5-CAACGTATGATTTAATTCCACC-3. PCR was performed
using KOD plus polymerase (Toyobo, Osaka, Japan)
under the following conditions: 94C for 2 min followed
by 30 cycles of denaturation at 94C for 30 s, annealing
at 50C for 30 s and polymerization at 68C for 30 s. All
PCR were performed in a T-GRADIENT thermocycler
(Biometra, Gottingen, Germany). The PCR products were
purified by Qiagen PCR purification kit (West Sussex,
UK), ligated into pGEM-T vector (Promega, Madison,
WI, USA) and subjected to DNA sequencing.
Bacteriocin purification
Cerein MRX1 was produced in a 1-l culture of TSBYE
(tryptic soy broth plus 06% yeast extract, Becton Dickinson) inoculated with 10 ml of strain MRX1 precultured for
24 h at 30C with shaking. The 1-l culture was carried out
in a 3-l Erlenmeyer flask for 20 h with shaking.
Subsequently, cells were harvested by centrifugation
(10 000 g, 15 min, 4C) and the antimicrobial activity was
concentrated by hydrophobic interaction chromatography
(HIC) as follows: the supernatant fluid was shaken with
20 g of Amberlite XAD-16 resin (Sigma, St Louis, MO,
USA) for 3 h. The matrix was then packed into a column
(25 by 20 cm, Bio-Rad, Tokyo, Japan) and washed successively with 100 ml of distilled H2O and 100 ml of 40%
ethanol. The bacteriocin was eluted with 200 ml of 70%
2-propanol containing 01% TFA (trifluoroacetic acid).
2-propanol was evaporated at 35C using a vacuum system.
The resulting material was diluted in 100 ml of sodium
phosphate buffer, 20 mmol l)1, pH 57 and subjected to a
20-ml SP-Sepharose Fast Flow cation-exchanger (15 by
14 cm, Amersham Pharmacia Biotech, Uppsala, Sweden)
at a flow rate of 2 ml min)1, using a peristaltic pump
(Micro Tube Pump MP-3, Eyela, Tokyo, Japan). Ten-ml
Fractions were collected during elution of the column with
a stepwise gradient from 0 to 1 mol l)1 NaCl. The active
fraction was diluted with 01% TFA and loaded into a

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S. Sebei et al.

Table 1 Culture conditions of bacteria and

inhibitory spectrum of cerein MRX1 as determined by the spot-on-lawn method

Purification and characterization of cerein MRX1

Indicator strain*

Medium

Temperature (C)

Inhibition

Bacillus cereus MRX1

Bacillus cereus DSM 345
Bacillus cereus DSM 487
Bacillus cereus JCM 2152T
Bacillus cereus CECT 5148
Bacillus thuringiensis CIP Bt7
Bacillus thuringiensis CIP Bt13
Bacillus mycoides DSM 2048T
Bacillus weihenstephanensis WBC 10204T
Bacillus badius IFR69
Bacillus circulans JCM 2504T
Bacillus circulans RBT18
Bacillus licheniformis RBTC
Bacillus megaterium FES333
Bacillus megaterium 68
Bacillus coagulans JCM 2257T
Bacillus subtilis JCM 1465T
Bacillus subtilis 65
Bacillus pumilus 38
Bacillus pumilus 24
Bacillus pumilus BP63
Bacillus pumilus BXX
Bacillus maroccanus CCM 6719
Paenibacillus alvei IFR11
Paenibacillus polymyxa CCM 1459
Paenibacillus macerans MR5
Virgibacillus pantothenticus IFR70
Brevibacillus laterosporus MJ18
Brevibacillus brevis FES434
Clostridium saccharoperbutylacetonicum
ATCC 13 564
Clostridium acetobutylicum IFO 13948
Listeria innocua ATCC 33090T
Micrococcus luteus IFO 12708
Leuconostoc mesenteroides
subsp. mesenteroides JCM 6124T
Enterococcus faecium TUA 1344 L
Lactococcus lactis subsp. lactis ATCC 19435T
Enterococcus faecalis JCM 5803T
Pediococcus pentosaceus JCM 5885
Lactobacillus sakei subsp. sakei JCM 1157T
Lactobacillus plantarum ATCC 14917T
Staphylococcus aureus subsp. aureus
ATCC 12600T
Staphylococcus epidermidis JCM 2414T
Streptococcus bovis JCM 5802T
Escherichia coli JM 109
Pseudomonas oleavorans ATCC 29347

TSBYE
TSBYE
TSBYE
TSBYE
TSBYE
TSB
TSB
TSB
TSB
TSB
TSBYE
TSB
TSB
TSB
TSB
TSBYE
TSBYE
TSBYE
TSB
TSB
TSB
TSB
TSB
TSB
TSB
TSBYE
TSBYE
TSB
TSB
RCM

30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
37
30
30
30
30
30
30
30
37
30
37
37
30
30
30

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
)
)
+
)
+

RCM
BHI
TSBYE
MRS

37
37
30
30

+
+

MRS
MRS
MRS
MRS
MRS
MRS
TSBYE

30
30
37
37
30
30
37

)
)
)
)
)
)
)

TSBYE
TSBYE
TSB
TSB

37
37
37
30

+
+
)
)

The active peak of cerein MRX1 shown in Fig. 1 was used to determine this spectrum.
*ATCC, American Type Culture Collection; CIP, Collection de lInstitut Pasteur, France; CECT,
Spanish Type Culture Collection; CCM, Crechoslovack Collection of Micro-organisms; DSM,
Deutsche Sammlung von Mikro-organismen; IFO, Institute for Fermentation, Osaka, Japan; JCM,
Japanese Collection of Micro-organisms; WBC, Weihenstephan Bacillus collection, Germany.
E. coli JM109 was from Promega. Others strains were laboratory isolates.
TSB, tryptic soy broth; TSBYE, TSB with 06% yeast extract; BHI, brain heart infusion; RCM,
reinforced clostridial medium; MRS, de Man-Rogosa-Sharpe agar. +, Strong inhibition of the
indicator strain; ), no inhibition; , hazy zone of inhibition.

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Purification and characterization of cerein MRX1

reversed-phase column (Resource RPC, Amersham Pharmacia Biotech) integrated in an LC-10A HPLC system
(Shimadzu, Kyoto, Japan). The column was eluted with a
linear gradient from 0% to 100% acetonitrile containing
01% TFA at a flow rate of 1 ml min)1. The bacterial cells
harvested from the 1-l culture were washed in 100 ml of
10 mmol l)1 sodium phosphate buffer (pH 68) for
15 min, and then resuspended in 20 ml of 01% TFA in
40% acetonitrile. The cell suspension was shaken gently for
15 min, then the cells were discarded by centrifugation
(14 000 g, 15 min, 4C) and the cell surfaces extract (containing the bacteriocin) passed through a syringe filter and
stored at )20C. Fractions obtained during all the purification procedures were assayed for antimicrobial activity by
the spot-on-lawn method (Tagg et al. 1976) using Bacillus
coagulans JCM 2257T as indicator organism. Protein concentration was estimated by the dye binding method
(Smith 1995).
Assay of bacteriocin activity
Bacteriocin activity was assayed by the critical dilution
method. Plates containing solid medium were overlaid
with 5 ml of soft agar lawns containing approx. 5 106
cells of the indicator strain. Portions (10 ll) of serial
twofold dilutions of bacteriocin solutions were spotted
onto these lawns. After incubation at the optimal conditions, the bacterial lawns were examined for growth
inhibitory zones. One unit of antimicrobial activity was
defined as the smallest amount to give a clear zone of
growth inhibition and the reciprocal of the highest dilution which caused such a zone was defined as the number
of antimicrobial units (AU) per millilitre. When the aim
of the experiment was to detect the presence of antimicrobial activity, 10 ll of the solutions was spotted on
the lawns without diluting (spot-on-lawn method).
Direct detection of bacteriocin activity
in polyacrylamide gel
SDSPAGE was curried out on a 16% gel with tricine as
trailing ion (Schagger and von Jagow 1987). The bacteriocin was loaded in duplicate onto the gel. After electrophoresis, the gel was cut vertically. One part of the gel
contained the peptide marker and cerein MRX1, the other
part contained cerein MRX1 only. The former part was
stained to visualize protein bands. For in situ detection of
bacteriocin activity (Bhunia et al. 1987), the second part
of the gel was fixed in a mixture of 2-propanol, acetic acid
and H2O (25 : 10 : 65) for 15 min and rinsed with sterile
H2O for 30 min. The gel was then placed in a Petri dish
and overlaid with 10 ml of soft TSBYE agar containing
100 ll of an overnight culture of B. coagulans JCM 2257T.
1624

S. Sebei et al.

After incubation overnight at 37C, the gel was examined

for the presence of growth inhibition zones. Finally, both
parts of the gel were rejoined and photographed.
Physicochemical properties
To test for the sensitivity to hydrolytic enzymes, aliquots
of the bacteriocin (20 lg ml)1) were treated for 4 h with
enzymes at a final concentration of 04 mg ml)1. Enzymes
(proteinase K, actinase E, a-chymotrypsin, a-amylase and
lipase) were dissolved in buffers as recommended by the
supplier (Sigma). Untreated bacteriocin in buffers was the
positive control, buffers alone and enzyme solutions were
the negative controls. To study its thermostability, cerein
MRX1 was incubated at various temperatures (40100C)
for 20 min or treated by autoclaving (121C, 15 min).
The pH stability was determined by storing the bacteriocin at 4C for 24 h in the following buffers: 01% TFA,
pH 20; 50 mmol l)1 acetate buffer, pH 3650;
50 mmol l)1 Tris-HCl buffer, pH 80 and pH 91. After
each treatment, residual activity was assayed against
B. coagulans JCM 2257T. Solubility in various solvents
was estimated by adding the solvents to tubes containing
lyophilized cerein MRX1 to reach final peptide concentrations ranging from 01 to 2 lg ll)1. Each tube was then
checked for the presence of precipitate. Stability in
organic solvents was assessed by mass spectrometry (MS)
after incubating these same tubes at 25C for 3 days.
Minimal inhibitory concentrations
Minimal inhibitory concentrations (MICs) were determined based on the method of Parrot et al. (1989) with
some modifications. Each tested strain was first grown to
exponential phase in its suitable culture conditions. The
culture was then diluted in the same fresh medium to a
final OD590 equal to about 04. This culture (100 ll) was
poured into each well of a microtiter plate (Falcon, Becton
Dickinson). Afterwards, twofold dilutions of the bacteriocin were freshly prepared and 25 ll of each dilution was
added to the wells to get a final volume of 125 ll. For the
positive control, 25 ll of fresh medium was added to one
well. Sterile fresh medium (125 ll) was poured into
another empty well to serve as a blank for a microplate reader (ImmunoMini NJ-2003, InterMed, Tokyo, Japan). The
plates were incubated until the tested strain grown without
bacteriocin reached stationary phase. The ODs in the wells
were measured at 590 nm using the microplate reader. The
ODs were plotted against the bacteriocin concentration
and the MIC was deduced from the obtained curve. MIC
was defined as the minimal bacteriocin concentration
which produced at least 50% reduction in the final growth
of the culture.

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S. Sebei et al.

Purification and characterization of cerein MRX1

Mass spectrometry and protein sequencing

Molecular masses were determined by matrix-assisted
laser desorption ionization time-of-flight (MALDI-TOF)
analysis using a Voyager RP mass spectrometer (Applied
Biosystems, Foster City, CA, USA). The matrix, a-cyano4-hydroxycinnamic acid (Sigma), was dissolved at a
concentration of 10 lg ll)1 in a mixture of 1 : 1 acetonitrile H2O containing 01% TFA. Afterwards, 1 ll of
sample solution and 1 ll of matrix were mixed together
by pipetting and 1 ll of the mixture was spotted on the
sample plate and air dried. All data were calibrated by an
external calibration standard mixture (Applied Biosystems). Alternatively, masses were determined by electrospray ionization time-of-flight MS (ESI-TOF MS) using a
JMS-T100LC mass spectrometer (JEOL, Tokyo, Japan).
N-terminal amino acid sequencing analyses were carried
out by Edman degradation on a PSQ-1 gas-phase automatic sequence analyser (Shimadzu).

Speedvac evaporator (Savant, Farmingdale, NY, USA).

Treatment by 2-mercaptoethanol was carried out according
to Meyer et al. (1994). Reduction and desulfurization was
carried out by a mixture of sodium borohydride and nickel
chloride according to Kawulka et al. (2004). After chemical
treatments, modified cerein MRX1 was purified from the
reaction mixtures by RP-HPLC.
Results
Identification of a bacteriocin-producing bacterium

Amino acid composition analysis was performed on an

automated amino acid analyser (JLC-500, JEOL) after
hydrolysis with 6 mol l)1 HCl containing 02% phenol at
110C for 24 h. Before use, cerein MRX1 was rechromatographed by RP-HPLC to remove trace amounts of
impurities and about 400 lg of peptide were used for the
analysis. This experiment was carried out twice. NMR
(nuclear magnetic resonance) spectroscopy was performed
with a Unityinova spectrometer (Varian, Palo Alto, CA,
USA) operating at a proton frequency of 600 MHz.
Proton spectra were measured at 25C with 5 mmol l)1
cerein MRX1 in CD3OD (methanol-d4).

Two hundred and seven newly isolated Bacillus strains were

screened for the production of bacteriocin by the method of
inverted agar (Tagg et al. 1976). Strain MRX1, isolated from
aquatic plants roots, strongly inhibited the growth of many
indicator bacteria including B. coagulans JCM 2257T and
Listeria innocua ATCC 33090T. The antimicrobial activity,
called cerein MRX1, was detected in the culture supernatant
collected during the stationary growth phase of the producer organism. Treatment of this supernatant by proteinase
K (1 mg ml)1) caused a complete inactivation of cerein
MRX1. This showed the proteinaceous nature of cerein
MRX1, which can be considered a bacteriocin. Cells of
strain MRX1 were Gram-positive, rod-shaped, catalase-positive, oxidase-positive and able to produce endospores (one
per cell). On API CHB galleries, strain MRX1 displayed the
typical fermentation profile of B. cereus and its closely related species B. mycoides. The 16S rDNA sequence of strain
MRX1 shared the highest level of similarity with the
sequence of B. cereus ATCC 25 621 (100% identity). In
addition, its gyrB gene sequence shared the highest level of
similarity with the sequence of B. cereus H16 (100% identity). Thus, the strain was named B. cereus MRX1.

Other methods

Purification of cerein MRX1

The mode of action of cerein MRX1 against B. coagulans

JCM 2257T was determined as previously described (Oscariz and Pisabarro 2000). For partial hydrolysis, lyophilized
bacteriocin (1 mg) was treated by 200 ll of 6 mol l)1 HCl
at 43C for 48 h. After incubation, the tube was centrifuged
(10 000 g, 5 min) to remove insoluble matter and the
excess HCl was removed by drying the solution in a

The bacteriocin was purified from the supernatant fluid of

a stationary phase culture of B. cereus MRX1, grown at
30C in TSBYE. HIC was used efficiently to concentrate the
antimicrobial activity from the supernatant. Subsequently,
933% recovery and more than twofold increase in specific
activity were obtained in this step. However, cationexchange chromatography (CEX) resulted in a significant

Amino acid analysis and NMR spectroscopy

Table 2 Purification of cerein MRX1 from the culture supernatant

Purification step

Volume
(ml)

Total protein
(mg)

Total activity
(AU)

Specific activity
(102 AU mg)1)

Recovery (%)

Purification
(fold)

Supernatant
HIC
CEX
RP-HPLC

1000
200
10
1

1246
4476
805
0076

300
280
16
12

240
625
1987
1578

100
933
53
40

10
26
82
656

000
000
000
000

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Purification and characterization of cerein MRX1

S. Sebei et al.

loss of activity (Table 2). The final separation step by

reversed-phase HPLC yielded one peak active against B. coagulans JCM 2257T. Cerein MRX1 showed absorbances at
220 and 280 nm suggesting the presence of a peptide with
aromatic residue(s). Its retention time was 24 min (Fig. 1).
When the HPLC active fraction was subjected to SDS
PAGE, it showed a single band that migrated at a position
that corresponded to a molecular mass of approx. 3 kDa as
can be seen in part (a) of Fig. 2. To this band was associated antimicrobial activity as can be noticed in part (b) of
the same figure. In preliminary studies on the production
of cerein MRX1 in batch cultures, we found that an
important proportion of the peptide was naturally bound
to the cell surfaces of the producer organism (data not
shown). We took advantage of this finding to design a secmAbs
2000

220 nm

1500
1000
500
0
1000

280 nm

500
0
0

10

20

30 min

Figure 1 Final step in the purification of cerein MRX1 by RP-HPLC.

UV absorption was registered at 220 and 280 nm. Elution of the
Resource RPC column was carried out by a linear gradient from 0%
to 100% of 01% TFA in acetonitrile for 30 min. The peak with antimicrobial activity is indicated by a horizontal bold line.

(a)
1

ond simplified procedure for large-amount purification of

the bacteriocin (see bacteriocin purification in section
Materials and methods). When 2 ml of the 20-ml cell
extract was diluted in 01% TFA, and injected onto the
Resource RPC column, cerein MRX1 could be easily separated from the other constituents and about 200 lg of chromatographically pure bacteriocin was usually obtained.
This means that 2 mg of cerein MRX1 was purified from
the cells of a 1-l bacterial culture while only 76 lg was purified from the supernatant fluid of the same culture. The
ability of cerein MRX1 to adsorb to cellular surfaces was
probably because of its high hydrophobicity.
Antimicrobial spectrum
The ability of the bacteriocin to inhibit the growth of various bacterial strains was tested on agar plate by the spoton-lawn method. As shown in Table 1, cerein MRX1 was
not clearly active against the producer strain itself but it
was active against other B. cereus strains. Cerein MRX1
inhibited species genetically related to the producer
organism (B. mycoides, B. thuringiensis and B. weihenstephanensis) and other Bacillus species such as B. coagulans,
B. circulans and B. megaterium. The inhibitory spectrum
encompassed many spore-formers such as Paenibacillus
and Clostridium. Other Gram-positive bacteria such as
Listeria innocua and Streptococcus bovis were also inhibited. Interestingly, the lactic acid bacteria: Leuconostoc
mesenteroides subsp. mesenteroides JCM 6124T, Enterococcus faecium TUA 1344 L, Lactococcus lactis subsp. lactis
ATCC 19435T, Enterococcus faecalis JCM 5803T, Pediococcus pentosaceus JCM 5885, Lactobacillus sakei subsp. sakei
JCM 1157T and Lactobacillus plantarum ATCC 14917T
were resistant to cerein MRX1. Escherichia coli and Pseudomonas oleavorans were among resistant bacteria.

(b)
2

MICs and mode of action

kDa
169
106
62
34

Figure 2 (a) SDSPAGE of purified cerein MRX1. Line 1, size marker;

line 2, band corresponding to the bacteriocin. (b) Gel containing
cerein MRX1 and overlaid with the indicator strain B. coagulans JCM
2257T. Line 1, inhibition zone caused by the bacteriocin.

1626

MICs of cerein MRX1 (in liquid media) against various

strains are shown in Table 3. This bacteriocin was particularly active against Bacillus strains and L. innocua (lowest
MICs). In contrast, lactic acid bacteria were resistant
(Ped. pentosaceus JCM 5885, Leuc. mesenteroides subsp.
mesenteroides JCM 6124T) or only inhibited by relatively
high concentrations of bacteriocin (Ent. faecalis JCM
5803T, Ent. faecium TUA 1344 L and Strep. bovis JCM
5802T). Like most bacteriocins produced by Gram-positive bacteria, cerein MRX1 was inactive against the
Gram-negative bacterium E. coli even at relatively high
concentrations. Cerein MRX1 showed a bactericidal mode
of action. In fact, addition of this bacteriocin (at a final
concentration of 50 AU ml)1) to an exponentially
growing culture of B. coagulans JCM 2257T caused an

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S. Sebei et al.

Purification and characterization of cerein MRX1

Table 3 MICs of cerein MRX1

Sensitive to low peptide concentration

Bacillus cereus DSM 345
Bacillus cereus JCM 2152T
Bacillus megaterium 68
Bacillus circulans JCM 2504T
Bacillus subtlis JCM 1465T
Bacillus pumilus BP63
Bacillus coagulans JCM 2257T
Listeria innocua ATTC 33090T
Resistant or sensitive to relatively high concentrations
Bacillus cereus MRX1
Enterococcus faecalis JCM 5803T
Enterococcus faecium TUA 1344 L
Streptococcus bovis JCM 5802T
Pediococcus pentosaceus JCM 5885
Leuconostoc mesenteroides
subsp. mesenteroides JCM 6124T
Micrococcus luteus IFO 12 708
Escherichia coli JM 109

MICs
(lg ml)1)

313991
4000

07
09
2
15
35
11
12
08
21
142
285
248
R
R
R
R

R, resistant to the highest peptide concentration tested (57 lg ml)1).

immediate decrease in viable cells number (compared

with a control culture without bacteriocin).
Physicochemical properties
Cerein MRX1 was not inactivated by a-amylase and lipase
but was completely inactivated by the proteases: proteinase
K, actinase E and a-chymotrypsin. This bacteriocin can be
considered very thermostable because its activity was not
affected by all temperatures tested, except autoclaving
which caused a relatively small decrease in antimicrobial
activity (20%). Besides, cerein MRX1 maintained its full
activity over a wide pH range (pH 3680). It was slightly
affected by extreme pHs (pH 20 and pH 91). At room
temperature, the bacteriocin was well soluble (i.e. completely soluble at all the concentrations tested) in methanol,
ethanol and 2-propanol. It was insoluble in chloroform
and acetonitrile and relatively soluble in H2O and acetonitrile containing 01% TFA (up to 05 and 01 lg ll)1,
respectively). The tubes containing cerein MRX1 dissolved
in methanol, acetonitrile with 01% TFA, ethanol and
2-propanol were incubated for 3 days at 25C. Possible
changes in the structure of the peptide were assessed by
MS. The bacteriocin remained stable in all these solvents
and no significant degradation could be noticed.
Molecular characterization
MALDI-TOF MS analysis of HPLC-purified cerein MRX1
gave a unique intense signal at m z = 313991 corres-

Intensity

Indicator strains

2000

0
3000

4000

m/z
Figure 3 MALD-TOF mass spectrometry analysis of cerein MRX1. The
m z value of 313991 corresponding to [M + H]+ indicates a molecular mass of 313891 Da.

ponding to [M + H]+ (Fig. 3). The monoisotopic mass of

cerein MRX1 calculated from the average of ten independent measures by ESI-TOF MS using a JEOL JMST100LC mass spectrometer was equal to 313793 Da.
Sequencing of the bacteriocin from its N-terminal end
was attempted using Edman degradation. The experiment
was repeated three times, but no conclusive sequence
could be determined. Cerein MRX1 was reduced by the
2-mercaptoethanol modification mixture of Meyer et al.
(1994) and purified by RP-HPLC with a gradient from 0
to 100% acetonitrile over 30 min. The retention time of
the reduced peptide was 19 min. Amino acid sequencing
revealed the partial sequence of the peptide as follows:
DWTXWSXLVXAAXSVELX (X at positions 4, 7, 10, 13
and 18 corresponds to unidentified residues). After partial
hydrolysis of cerein MRX1 in HCl, a fragment having the
mass of 58146 Da was isolated by RP-HPLC. Its sequence
was found to be SVELL (corresponds to residues 1418).
A second isolated fragment with a mass of 91472 Da gave
the N-terminal sequence: SVEL, after which sequencing
stopped. The rest of this fragment corresponds to a mass
of 48624 Da. The blockage of sequencing suggested the
involvement of the residues next to leucine 18 (probably
residue 19) in a post-translational modification.
Amino acid composition and classification
The reducing and desulfurizing agent nickel boride (which
forms in the mixture of sodium borohydride and nickel
chloride) was previously used to convert cysteine residues
into alanine residues in the case of the lantibiotic lacticin
3147 (Martin et al. 2004) and the bacteriocin, subtilosin A

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Purification and characterization of cerein MRX1

S. Sebei et al.

Table 4 Amino acid composition analysis of cerein MRX1

Amino acid residues

Residue per molecule*

Ala
Asx
Cys
Glx
Gly
Leu
Ser
Thr
Trp
Val

6
1
1
1
1
4
3
2
0
3

(586)
(085)
(076)
(096)
(103)
(400)
(218)
(158)
(000)
(283)

*In parenthesis are the experimental values determined by an automated amino acid analyser after hydrolysis of the peptide with 6 mol l)1
HCl for 24 h at 110C. Under these conditions hydroxy-substituted
residues are partially destroyed and Trp is degraded. The determination of hydroxy-substituted residues takes into account the fact that
the ratios observed for Ser (here 218) and Thr (here 158) are typically
30% and 13% below the real values, respectively (Dunn 1995).
From the Edman sequencing data, it was clear that residues Asx and
Glx correspond to 1 Asp and 1 Glu, respectively.
In addition to the 1 Cys residue determined by the amino acid analysis, Edman sequencing showed the presence of 3 other cysteines.

(Kawulka et al. 2004). Nickel boride-treated cerein MRX1

was subjected to Edman sequencing and the following
sequence was obtained: DWTAWSALVAAAASVELL. This
result clearly indicated that the unidentified residues at
positions 4, 7, 10 and 13 were cysteines. Therefore, the
N-terminal part of cerein MRX1 was suggested to be the
following: DWTCWSCLVCAACSVELL. Analysis of amino
acid composition after acid hydrolysis indicated that the
bacteriocin consisted of usual amino acids found in
ribosomally synthesized peptides (Table 4). Notably,
lanthionine was absent. Thus, cerein MRX1 is not a lantibiotic and can be classified as a class II bacteriocin. This
classification was confirmed by NMR as explained in
Fig. 4. Taking into account both the amino acid analysis
and the data of Edman degradation sequencing, 27 amino
acid residues in cerein MRX1 were so far determined: 6
Ala, 1 Asp, 4 Cys, 1 Glu, 1 Gly, 4 Leu, 3 Ser, 2 Thr, 2 Trp
and 3 Val. The calculated total mass of these 27 residues
is 274421 Da, which is lower than the measured mass
of the bacteriocin by 39372 Da. This indicates that
other residues (the most probably three) were not yet
determined.
Discussion
During the last decade, the incidence of microbial resistance to conventional antibiotics has increased dramatically (Okeke et al. 2005). In addition, consumers are
no longer satisfied with the use of potentially harmful
chemical preservatives in foodstuffs and other processed
1628

Aromatic protons region

Vinyl protons region

(ppm)
Figure 4 One-dimensional NMR spectrum of cerein MRX1. The part
of the spectrum that encompasses the vinyl and aromatic regions is
shown. The absence of vinyl protons which give doublet [because of
dehydroalanine (Dha)] or quartet [because of dehydrobutyrine (Dhb)]
resonance peaks in the d = 5269 ppm region of the spectrum
(Jones et al. 1983; Liu and Hansen 1992; Paik et al. 1998) is
an important indicator for the absence of the dehydro amino acids
Dha and Dhb. Thus, cerein MRX1 is not a lantibiotic but a class II
bacteriocin.

products. Thus, the need to discover novel antimicrobials

is greater than ever. Among the newly emerging tools to
fight undesirable micro-organisms, bacteriocins produced
by Gram-positive bacteria are considered to be the most
promising for applications in the near future. Here, we
reported about a novel bacteriocin, cerein MRX1, produced by a new isolate: B. cereus MRX1. The molecular
mass of cerein MRX1 as determined by MS was
313793 Da. This value is in the mass range of most class
I and class II bacteriocins. Cerein MRX1 had the ability
to inhibit many spoilage bacteria (Bacillus species,
L. innocua) at relatively low concentrations (MICs in the
range of 1 lg ml)1) that were not inhibitory to useful
lactic acid bacteria (e.g. Ped. pentosaceus). This property
of cerein MRX1 may give an advantage to its application
as an antibacterial agent.
In order to study the biological activities and chemical
structures of bacteriocins, it is necessary to develop
appropriate purification procedures. Bacteriocins produced by Gram-positive bacteria are the most often secreted into the growth medium. Therefore, the purification
preferably starts by a concentration step using the cell-free
culture supernatant. Then, several additional steps, the
most often chromatographic, are necessary to achieve a
significant purity. Purification of cerein MRX1 from the
supernatant was carried out by three chromatographic
steps, namely hydrophobic interaction, cation-exchange
and reversed-phase high performance liquid chromatographies. The retention time of cerein MRX1 on the
reversed-phase column (last protein eluted at 24 min,
corresponding to 80% acetonitrile) indicated its high
hydrophobicity. CEX resulted in a low recovery. This was
probably because of a weak retention of the bacteriocin

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S. Sebei et al.

on SP-Sepharose, leading to a loss of activity during sample loading and column washing (before elution by
NaCl). The amino acid composition supports this explanation. Cerein MRX1 contains acidic residues (1 Asp and 1
Glu) but not basic residues (Arg, Lys and His). Therefore,
this bacteriocin is expected to have a net negative charge
(close to )2) over a relatively wide range of pH. It is
important to notice that this feature is rare among bacteriocins from Gram-positive bacteria. Most bacteriocins
are cationic because they contain an excess of basic residues (Jack et al. 1995). Like some bacteriocins produced
by lactic acid bacteria (Hastings et al. 1991; Jack et al.
1995), cerein MRX1 was found both in a cell-associated
form and in the culture supernatant fluid of the producer
organism. This property of some bacteriocins to be able
to adsorb on the cellular surfaces was previously used to
design efficient adsorption desorption methods for their
recovery on a large scale. For example, the adsorption on
cells of nisin and pediocin AcH secreted in liquid media
could be promoted by adjusting the pH (of the same
media in which the strains had already grown) to 60.
The adsorption step could then be followed by cell separation and bacteriocin desorption at pH 20 (Yang et al.
1992; Daba et al. 1994). Even if inspired by those earlier
procedures, our way of cerein MRX1 recovery from the
cell surfaces was different and simpler. In fact, a relatively
large amount of cerein MRX1 was naturally adsorbed on
the cells of strain MRX1. An adsorption step by pH
adjustment was not required.
Although there are many scientific reports about the
production of bacteriocins or bacteriocin-like substances
by strains of B. cereus, purification, N-terminal amino acid
sequencing and accurate molecular mass determination
were only carried out for cerein 7A (Edman sequencing
yielded: GWGDVL), cerein 7B (Edman sequencing yielded:
GWWNSWGH) and in this work, for cerein MRX1
(N-terminus: DWTCWSCLVCAACSVELL). Both cereins
7A and 7B were isolated from B. cereus Bc7 (Oscariz et al.
2006). The determined 18-amino acid sequence of cerein
MRX1 is different from the sequences of cereins 7A and
7B but very similar to the 18-amino acid sequence of a
recently discovered 3162-Da bacteriocin, thuricin 17 produced by B. thuringiensis NEB17 (Gray et al. 2006). The
difference between the two sequences resides in the residue
in position 10 which is a cysteine in cerein MRX1 while it
is a valine in thuricin 17. A noticeable similarity between
the three cereins and thuricin 17 is the presence of one or
more tryptophan residues in their N-terminal region. Particularly, the conserved tryptophan in position 2 probably
plays an important role in the biological activity of
these four bacteriocins. Another marked similarity between
cerein MRX1 and thuricin 17 is the blockage of Edman
sequencing at the same position. This leads us to think

Purification and characterization of cerein MRX1

that the two bacteriocins have similar structures (i.e.

amino acid sequence and intramolecular linkages). The
blockage of sequencing suggests that residue 19 is probably
implicated in an unusual post-translational modification.
Cerein MRX1 and thuricin 17 are different with regard to
their molecular masses and amino acid compositions
(Gray et al. 2006). The evidence that cerein MRX1 is a
class II bacteriocin is strong. In fact, lanthionine and
dehydro amino acid residues (Dha and Dhb), characteristically present in class I bacteriocins, were absent in cerein
MRX1. This bacteriocin does not belong either to the subclass IIa (because of the absence of the conserved N-terminal motif YGNGV) or the subclass IIb of bacteriocins
(because it is not a two-peptide bacteriocin). Finally, it
does not belong to the subclass IIc (circular bacteriocins)
because reduced cerein MRX1 could be sequenced from its
N-terminus. Altogether, these data are in favour of cerein
MRX1 belonging to Cotter and co-workers subclass IId of
bacteriocins. Currently, two-dimensional 1H-NMR spectra
of cerein MRX1 are being analysed in order to determine
its complete structure.
Acknowledgements
This study was financially supported by the Ministry of
Education, Culture, Sports, Science and Technology,
Japan. We gratefully thank Dr Koji Nagata (Laboratory of
Food Biotechnology, Department of Applied Biological
Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo) for his kind help with
NMR spectroscopy. We also gratefully thank Mr Kiyoshi
Ogawa (Asahi Kasei Pharma) for his help with the amino
acid analyses.
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