Báo cáo khóa học: Purification and functional characterization of insecticidal sphingomyelinase C produced by Bacillus cereus ppt

Purification and functional characterization of insecticidal
sphingomyelinase C produced by
Bacillus cereus
Hisashi Nishiwaki, Katsuhiko Ito, Katsuhiko Otsuki, Hiroyuki Yamamoto, Koichiro Komai
and Kazuhiko Matsuda
Department of Agricultural Chemistry, Faculty of Agriculture, Kinki University, Nara, Japan
Bacillus cereus isolated from the larvae of Myrmeleon bore
was found to secrete proteins that paralyze and kill German
cockroaches, Blattela germanica, when injected. One of
these active proteins was purified from the culture broth
of B. cereus using anion-exchange and gel-filtration chro-
matography. The purified toxin, with a molecular mass of
34 kDa, was identified as sphingomyelinase C (EC 3.1.4.12)
on the basis of its N-terminal and internal amino-acid
sequences. A recombinant sphingomyelinase C expressed in
Escherichia coli was as potent as the native protein in killing
the cockroaches. Site-directed mutagenesis (His151Ala) that
inactivated the sphingomyelinase activity also abolished the
insecticidal activity, suggesting that the rapid insect toxicity
of sphingomyelinase C results from its phospholipid-
degrading activity.
Keywords: antlion; Bacillus cereus; insecticidal activity;
Myrmeleon bore; sphingomyelinase C.
A group of antlions, the larvae of lacewing Myrmeleonti-
dae, make pits to capture prey. Before sucking the body
fluid, antlions inject their regurgitant into the prey from
a pair of mandibles for extra digestion. As the prey of
antlions appear to be paralyzed, it has been postulated that
toxic factors are contained in the regurgitant. In preliminary
experiments, the insecticidal factors were found to be
sensitive to heat and proteinase treatments, indicating that

they are polypeptides. Antlion Myrmeleon bore toxin has
been purified from the regurgitant of larvae of M. bore and
shown to be a single polypeptide with a molecular mass of
170 kDa [1]. In addition to this toxin, a GroEL homolog
has recently been isolated as a toxic principle from the
culture broth of a symbiont, Enterobacter aerogenes [2].
Although the insecticidal activity of these proteins has
been evaluated, it is unclear whether the toxins in the
regurgitant of antlions are limited to these two proteins,
and whether other symbionts of the larvae also produce
insecticidal proteins that contribute to the toxicity of the
regurgitant.
In this study, Bacillus cereus was isolated from the larvae
of M. bore and found to produce insecticidal factors when
cultured aerobically. One of these was purified to homo-
geneity and tested for insecticidal activity by injecting it into
German cockroaches, Blattela germanica, and common
cutworms, Spodoptera litura. The active principle purified
from the bacterial culture broth was found to be sphingo-
myelinase C (SMC), which paralyzes the insects shortly
after injection. Recombinant SMC expressed in Escherichia
coli was as potent as the native protein. However, when
His151 was replaced by Ala, not only the phospholipid-
hydrolyzing rate but also the insecticidal activity of the
recombinant SMC was markedly reduced, suggesting a
close link between the insecticidal and enzyme activities.
Materials and methods
Insects
Last instars of antlions, M. bore, were collected in Tottori
prefecture, Japan and reared at 25 °C before use. Adult

German cockroaches, Blattela germanica, were kindly
provided by Sumitomo Chemical Co. Ltd (Hyogo, Japan).
Larvae of common cutworms, Spodoptera litura,were
purchased from Sumika Techno Service Co. Ltd (Hyogo,
Japan). Both insects were reared at 26 °Cand60%
humidity.
Injection assay
The injection assay with the cockroaches was conducted
as reported previously [1]. In brief, 2 lL culture broth or a
solution containing a protein sample was injected into the
abdomen of adult male German cockroaches. Five cock-
roaches were used for each dose of sample, and the
symptoms of the cockroaches were observed 10 min after
injection. The minimum paralysis dose (MPD, ng per insect)
at which at least four of five insects were paralyzed was
determined as the toxicity index of the sample. In the same
way, the MPD values for common cutworms was deter-
mined for the wild-type and mutant recombinant toxins
expressed in E. coli,after5lL of the protein solution was
injected into the side of the larvae.
Correspondence to K. Matsuda, Department of Agricultural
Chemistry, Faculty of Agriculture, Kinki University,
3327-204 Nakamachi, Nara 631-8505, Japan.
Fax: + 81 742 431445, Tel.: + 81 742 431511 extn 3306,
E-mail:
Abbreviations: KPB, potassium phosphate buffer; SMC,
sphingomyelinase C; MPD, minimum paralysis dose;
SCD, soybean casein digest.
Enzyme: sphingomyelinase C (EC 3.1.4.12).
(Received 20 October 2003, revised 3 December 2003,

accepted 10 December 2003)
Eur. J. Biochem. 271, 601–606 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2003.03962.x
Isolation of the bacteria from antlion
M. bore
and preparation of the culture broth
The antlions were sterilized with an aqueous 70% ethanol
solution, and one of the mandibles was pulled out using
sterilized forceps. Esophageal tissue and its contents were
streaked on soybean casein digest (SCD; pH  7.5; Nihon-
seiyaku Co. Ltd, Tokyo, Japan) agar plates, and the plates
incubated at 25 °C under aerobic conditions. After incuba-
tion for 1–2 days, bacteria that had grown on the plates
were added to 2 mL SCD liquid broth, and cultured at
25 °C for 16 h with shaking. This preculture was added to
2 mL fresh SCD broth at a final A
600
value of 0.05, and then
the second culture was shaken at 25 °C for 24 h. The culture
was centrifuged, and the supernatant filtered using a 0.2-lm
membrane filter (Millipore). The bacteria-free supernatant
was tested for its insecticidal activity against the cockroaches
by injection.
Identification of
B. cereus
To identify the bacterial species producing insecticidal
toxins, its 16S rRNA gene was amplified by PCR and
sequenced. The 16S rRNA gene was amplified using 1 U
KOD-Plus-polymerase (Toyobo Co. Ltd, Osaka, Japan),
15 pmol universal 16S rRNA gene primers (forward
primer, 5¢-AGAGTTTGATCCTGGCTCAG-3¢; reverse

primer, 5¢-GGCTACCTTGTTACGACTT-3¢), 1 m
M
MgSO
4
,0.2m
M
dNTP and 100 ng genomic DNA by
the following protocol (final volume, 50 lL): 94 °Cfor
2 min followed by 30 cycles of 94 °Cfor15s,50°Cfor
30 s, and 68 °C for 2 min. The amplified DNA band was
purified using low melting point agarose (Promega) and
cloned into pCRScpirt
TM
Amp SK(+) cloning vector
(Stratagene). The 16S rRNA gene was sequenced by the
dye-terminator method using DYEnamic ET Terminator
Cycle Sequencing Kit (Amersham Biosciences Co.,
Piscataway,
2
NJ, USA) combined with an ABI3100
Genetic Analyzer (Applied Biosystems Japan Ltd, Tokyo,
Japan). Biochemical properties of the bacterial species
were analyzed using API50 CHB and API20E test kits
(bioMe
´
rieux Japan, Tokyo, Japan).
Heat shock and proteinase treatments of the culture
broth of
B. cereus
To examine whether the toxic factors in the culture broth

of B. cereus were proteinous, effects of heat and prote-
inase treatments on the insecticidal activity of the
bacterial culture broth were examined. The filter-sterilized
supernatant (60 mL) of the culture broth of B. cereus
was concentrated to 600 lLusingamembranewitha
cut-off molecular mass of 10 kDa (Centriprep YM-10;
Millipore). Some of the solution was heated at 100 °C
for 10 min, and its toxicity tested by injection into the
cockroaches as described above. The rest of the sample
was treated with 0.1 mgÆmL
)1
proteinase K (Sigma-
Aldrich Japan K. K., Tokyo, Japan) at 0, 30, 60, 90 and
120 min in 25 m
M
potassium phosphate buffer (KPB,
pH 7.5) at 37 °C (final volume, 100 lL). At each time
point, the toxicity of the solution was tested by injection
into cockroaches.
Purification of a toxic protein produced by
B. cereus
B. cereus was cultured aerobically in three steps to prepare
a large volume of the broth, from which one of the toxic
factors was purified. The bacteria were cultured in 2 mL
SCD broth at 25 °C for 20 h, and an aliquot was added to
10 mL SCD broth (pH 8.0, initial A
600
0.05), which was
cultured at 25 °C for 6 h. Then the broth was added to
500 mL SCD medium (pH 8.0) at a final A

600
value of 0.05.
After the bacteria had been cultured with shaking for 8 h at
25 °C, the broth was centrifuged at 10 000 g for 20 min at
4 °C, and ammonium sulfate was added to the supernatant
to 40% saturation. The supernatant was left on ice for
60 min, and protein precipitates were removed by filtration
using a bottle top vacuum filtration system (Asahi Techno
Glass Co., Chiba, Japan). Ammonium sulfate was further
added to the filtrate to increase its concentration to 60%
saturation, and the solution was left on ice for 60 min. The
precipitates were harvested by centrifugation at 10 000 g for
30 min at 4 °C and dissolved in 7 mL 25 m
M
KPB (pH 7.5)
containing 1 m
M
dithiothreitol. After filtration using a
0.2-lm disposable syringe filter unit (Toyo Roshi Kaisha
Ltd, Tokyo, Japan) to remove insoluble substances, the
buffer containing ammonium sulfate was replaced with
25 m
M
KPB (pH 7.5) containing 1 m
M
dithiothreitol using
a HiPrep Desalting column (Amersham Biosciences) in an
A
¨
KTA prime system (Amersham Biosciences). Then the

protein solution was applied to an anion-exchange column
containing DEAE-Sepharose resin (HiPrep 16/10 DEAE;
Amersham Biosciences) using an A
¨
KTA explorer 10S
system. The column was washed with 200 mL 25 m
M
KPB
(pH 7.5) containing 1 m
M
dithiothreitol, and absorbed
proteins were eluted from the resin by increasing the KCl
concentration in KPB stepwise from zero to 150 m
M
and
500 m
M
in this order as shown in Fig. 1A. Ammonium
sulfate was added to all fractions so as to give a final
concentration of 80% saturation, and the solutions were left
on ice for 30 min. After centrifugation at 10 000 g for
30 min at 4 °C, each protein pellet was dissolved in 25 m
M
KPB (pH 7.5) containing 1 m
M
dithiothreitol. The fraction
eluted with the buffer containing 150 m
M
KCl was concen-
trated to 500 lL using a Centricon YM-10 (Millipore), and

the sample was fractionated by gel filtration using a
Superdex 200 column HR 10/300 (Amersham Biosciences)
with 25 m
M
KPB (pH 7.5) containing 1 m
M
dithiothreitol
at a flow rate of 0.5 mLÆmin
)1
. In all chromatographic
separations, proteins were detected by their absorbance at
280 nm. The protein concentration of each solution was
determined by the Bradford method [3]
4
using Coomassie
Plus-200 Protein Assay Reagent (Pierce) with BSA as the
standard. The purity of the active protein sample was
checked by 10% SDS/PAGE under reducing conditions by
the method of Laemmli [4], when the proteins were stained
with Coomassie Brilliant Blue R250 (Nacalai tesque Inc.,
Kyoto, Japan).
Sequencing of the proteinous toxin produced
by
B. cereus
The N-terminal and internal amino-acid sequences of the
purified toxic protein were analyzed by Edman degrada-
tion. The N-terminal amino-acid sequence was determined
602 H. Nishiwaki et al.(Eur. J. Biochem. 271) Ó FEBS 2004
after the protein sample had been blotted on a poly(viny-
lidene difluoride) membrane filter (Immobilon-P transfer

membrane; Millipore), and the internal amino-acid
sequence was determined by sequencing the N-terminal
part of a peptide fragment obtained by tryptic digestion of
the protein.
Cloning and sequencing of the insecticidal toxin (
SMC
)
gene of
B. cereus
The SMC-encoding gene was amplified by PCR using 1 U
KOD Plus polymerase, the primers (forward primer,
5¢-CAAATGGAGGTATGGAACG-3¢; reverse primer,
5¢-GCACAAGGTAATGGAACTTC-3¢), 1 m
M
MgSO
4
,
0.2 m
M
dNTP and 100 ng genomic DNA as template
according to the following protocol: 94 °Cfor2min
followed by 30 cycles of 94 °C for 15 s, 53 °Cfor30s,
and 68 °C for 1.5 min. The amplified gene was cloned into
pCRScpirt
TM
Amp SK(+) cloning vector. Then the cloned
gene was sequenced using the dye-terminator method with a
DYEnamic ET Terminator Cycle Sequencing Kit and
ABI3100 analyzer.
Functional expression of the insecticidal toxin (SMC)

in
E. coli
The SMC gene amplified by PCR using the vector sense
(VS) (5¢-GGGAATTCCATATGGAAGTGTCTACAA
ATC-3¢) and vector antisense (VA) (5¢-CCGCTCG
AGCTTCATAGAAATAGTCGCCTC-3¢)primerswas
cloned into NdeIandXhoI sites of pET22b(+) vector
(Novagen). The BL21[DE3]pLysS strain (Novagen) of
E. coli transformed with this expression vector containing
the SMC gene was incubated at 37 °C for 3 h, and protein
expression was then induced by addition of 1 m
M
isopropyl
thio-b-
D
-galactoside. After incubation at 25 °C for 14 h,
E. coli overexpressing the toxin was lysed with 10 mL
Bugbuster reagent (Novagen) containing 250 U Benzonase
Nuclease (Novagen). The supernatant of the bacterial lysate
was diluted twofold with 50 m
M
KPB (pH 7.5) and applied
to a Ni/nitrilotriacetic acid affinity column (Ni-NTA His-
Bind Resin; Novagen). The column was washed with
25 m
M
KPB (pH 7.5), and the absorbed protein was eluted
with 25 m
M
KPB (pH 7.5) containing 400 m

M
imidazole.
The eluted sample was further purified by gel filtration using
a Superdex 75 column 10/300 (Amersham Biosciences) with
25 m
M
KPB (pH 7.5).
Site-directed mutagenesis
The B. cereus SMC gene cloned in the pET22b expression
vectorwasusedasatemplateformutagenesis.The
mutation His151Ala (H151A) was introduced by PCR.
Mutagenesis sense (MS) 5¢-GGTACAGCGTTGCAA
GCGG-3¢ and mutagenesis antisense (MA) 5¢-CCGC
TTGCAACGCTGTACC-3¢ primers were designed to
generate the mutation. A pair of first-round PCRs was
carried out using 1 U KOD Plus polymerase, 100 ng wild-
type SMC gene cloned into pET22b(+) as template,
15 pmol of the primers (VS and MA, MS and VA), 1 m
M
MgSO
4
and 0.2 m
M
dNTP mixture in a 50-lL solution at
94 °C for 2 min followed by 30 cycles of 94 °Cfor15s,
50 °Cfor30s,and68°C for 1 min. The second-round
PCR was performed using 1 U KOD -Plus- polymerase,
50 ng each of the first-round PCR products, 15 pmol of the
primers (VS and VA), 1 m
M

MgSO
4
and 0.2 m
M
dNTP
mixture in a 50-lL solution at 94 °C for 2 min followed by
35 cycles of 94 °Cfor15s,50°Cfor30s,and68°Cfor
2 min, yielding a single band of predicted size. The DNA
band was digested with NdeIandXhoI, and cloned into the
NdeIandXhoI sites of pET22b. The H151A mutant protein
of SMC expressed in E. coli was purified using the protocol
described above.
Assay of sphingomyelinase activity
Sphingomyelinase activity was measured using an AmplexÒ
Red Sphingomyelinase Assay Kit (Molecular Probes). For
the measurement, the buffer was replaced with 100 m
M
Tris/HCl buffer (pH 7.5) using a Superdex 75 column 10/
300. To 5 lL 100 m
M
Tris/HCl buffer (pH 7.5) containing
sphingomyelin (200, 500, 1000, 2000, 3000 and 4000 l
M
)
and 2% Triton X-100 in each well of a 96-well microplate
(Optiplate
TM
96F; Packard Instrument Co.) was added
45 lL100m
M

Tris/HCl buffer (pH 7.5) containing 10 m
M
MgCl
2
,100l
M
Amplex red reagent, 2 UÆmL
)1
horseradish
peroxidase, 0.2 UÆmL
)1
choline oxidase and 8 UÆmL
)1
alkaline phosphatase, followed by 50 lL of the wild-type
or H151A mutant SMC (1 ngÆlL
)1
). After incubation for
30 min at 37 °C, the fluorescence at 590 nm was measured,
with excitation at 544 nm, using a microplate reader
(Wallac 1420 ARVOsx Malti label counter; Perkin–Elmer
Life Sciences, Tokyo, Japan). The dose–fluorescence
Fig. 1. Anion-exchange and gel-filtration chromatography profiles of the
insecticidal proteins produced by B. cereus. (A) After the DEAE-
Sepharose column had been washed with KPB containing 1 m
M
dithiothreitol, the proteins were eluted by increasing the KCl concen-
trationinKPBwith1m
M
dithiothreitol from zero to 150 m
M

and
500 m
M
in this order. (B) Gel filtration was conducted at a flow rate of
0.5 mLÆmin
)1
with KPB containing 1 m
M
dithiothreitol using a
Superdex 200 HR 10/300 column to give peaks i and ii in the profile.
The insecticidal SMC was found in peak ii.
Ó FEBS 2004 Insecticidal sphingomyelinase from B. cereus (Eur. J. Biochem. 271) 603
intensity data were fitted using
PRISM
software (Graphpad
software Inc., San Diego,
5
CA, USA).
Results and discussion
The SCD culture broth of a Gram-positive bacterial strain
isolated from the larvae of M. bore was found to rapidly
paralyze German cockroaches after injection. We examined
the 16S rRNA gene sequence and biochemical properties of
the bacterium. The 16S rRNA sequence was highly homo-
logous (99.9% identity) with that of B. cereus ATCC 14579
[5], and the biochemical profile obtained using the API tests
(see Materials and methods) agreed well with this sequen-
cing result. Therefore, we concluded that the bacterial
species producing insecticidal toxins isolated from the larvae
of M. bore is a strain of B. cereus.

The B. cereus strain has been isolated repeatedly from
the digestive system of the antlions M. bore (
6
H. Nishiwaki,
K. Ito, K. Nakashima, K. Fujiwara, M. Morimoto,
Y. Matsuda, H. Toyoda, K. Komai & K. Matsuda,
unpublished data), suggesting that the insecticidal factors
produced by B. cereus may aid the prey-capturing action of
the antlions. It is of interest that the bacterium was capable
of growing even at 50 °C, as antlions live in sandy ground
which may be very hot during the daytime in summer.
The insecticidal activity of the bacterial culture was
abolished not only by heating at 100 °C, but also by
proteinase K treatment (data not shown). In addition, the
insecticidal factors in the bacterial culture broth were not
removed by a membrane with a cut off molecular mass of
10 kDa, indicating that the active factors were polypeptides
larger than 10 kDa.
When purified using DEAE-Sepharose resin, the proteins
produced by B. cereus were separated into a ÔThroughÕ
fraction, which was not absorbed by the resin, and KCl-
eluted fractions, which were eluted by increasing the KCl
concentration in the buffer (Fig. 1A). The MPD value of
the Through fraction was 328 ± 194 (n ¼ 2, mean ±
SEM), whereas that of the fractions eluted by 150 m
M
KCl
and 500 m
M
KCl were 167 ± 63 (n ¼ 2) and > 642 (n ¼ 1),

respectively. It has been reported that phospholipase C of
B. cereus, which is not absorbed by DEAE-cellulose resin,
showed insecticidal activity [6,7]. Thus, to obtain other
insecticidal factors, we decided to purify the insecticidal
factors in the 150 m
M
KCl-eluted fraction by gel filtration.
The fraction eluted from DEAE-Sepharose by KPB
containing 150 m
M
KCl was separated into two major
peaks, i and ii, by gel filtration using the Superdex 200
column (Fig. 1B). An insecticidal protein with a molecular
mass of 70 kDa was purified from fraction i. However,
repeated isolation was difficult probably because of degra-
dation (data not shown). Another insecticidal protein was
also purified from fraction ii. This protein migrated as a
single band at a molecular mass of 34 kDa on electrophor-
esis on SDS/10% polyacrylamide (Fig. 2A; the yield of
protein was 142 ± 91 lg, mean ± SEM, n ¼ 2). The
34-kDa protein was able to intoxicate the cockroaches with
a MPD of 262 ± 29 ng protein/insect (mean ± SEM,
n ¼ 2). In addition to these two proteins, peaks i and ii
were also found to contain several other proteins. Although
these proteins may also exhibit insecticidal activity, we were
unable to evaluate it.
The N-terminal and internal amino-acid sequences
determined by Edman degradation of the 34-kDa insec-
ticidal protein were EVSTNQNDTLKVMTHNVYMLS
TNLYP and PQWTVTSWFQK, respectively. Both

sequences showed high homology with the sequence of
sphingomyelin phosphodiesterase (SMC) of B. cereus.To
confirm that the active principle is SMC, the SMC-
encoding gene was amplified by PCR from B. cereus,
sequenced, and functionally expressed by E. coli.The
SMC gene sequence (Fig. 3) cloned from B. cereus was
almost identical with that clarified by genome sequencing
of the B. cereus strain ATCC 14579 [5]. In addition, the
N-terminal and internal amino-acid sequences deduced
from the cloned gene were the same as those determined
by Edman degradation.
The recombinant protein expressed by E. coli was
purified homogeneously using the Ni/nitrilotriacetic acid
affinity column combined with the gel-filtration column
(Fig. 2B, W). The insecticidal activities (MPD, ng per
insect) of the recombinant SMC against the German
cockroaches and common cutworms were 161 ± 46
(n ¼ 3), a value close to that of the native protein, and
110 ± 10 (n ¼ 3), respectively. The fact that the recom-
binant and native proteins showed similar insecticidal
activity indicates that the insecticidal protein purified from
the culture broth of B. cereus is SMC. Heating at 50 °C
for 1 h markedly reduced the insecticidal activity of the
recombinant SMC (data not shown). Therefore, it is
conceivable that SMC is able to act as an exotoxin at lower
temperatures.
Several amino-acid residues involved in the sphingo-
myelinase activity of SMC have been identified [8–10]. It has
been proposed that His151 is involved in the hydrolytic
activity by interacting as a general acid with the phosphate

moiety in sphingomyelin. Therefore, we investigated the
effect of the H151A mutation on the insecticidal and
Fig. 2. SDS/PAGE of native SMC and the recombinant protein. SDS/
PAGE of native SMC purified from the culture broth of B. cereus (A;
10% gel) and the recombinant protein, which was expressed by E. coli
and subsequently purified by affinity and gel-filtration chromatogra-
phy [B; wild-type (W) and H151A mutant (M); 14% gel].
604 H. Nishiwaki et al.(Eur. J. Biochem. 271) Ó FEBS 2004
enzyme activity of recombinant SMC. As shown in Fig. 4,
the mutation markedly reduced the maximum rate of
hydrolysis of sphingomyelin, and the affinity of sphingo-
myelin for the wild-type and H151A mutant SMCs was
almost the same (K
d
 50 l
M
), suggesting that the catalytic
rate of SMC was reduced by the mutation. Concomitantly
with this decrease, the H151A mutation also abolished
the acute insecticidal activity [MPD value (ng per insect):
for cockroaches, > 860 (n ¼ 3); for cutworms, > 635
(n ¼ 3)], suggesting that the insecticidal activity of SMC
probably results from its sphingomyelinase activity.
Although it has been reported that invertebrates
contain little or no sphingomyelin in their tissues [11],
lysenin, a sphingomyelin-specific binding protein from the
coelomic fluid of the earthworm Eisenia foetida, has been
shown to affect the behavior of the spermatozoa of some
insects [12]. Thus, the tissue membranes of the insects
tested may contain sphinomyelin. Injection of SMC at

low doses ( 5 pmol per insect) resulted in loss of
mobility within a very short period. This implies that
the insecticidal effect of SMC is due to its action on the
nervous system. It will therefore be of interest to
determine if a selective action of SMC on the nervous
Fig. 3. Nucleotide sequence of the SMC gene
of B. cereus isolated from the larvae of M. bore
and its amino-acid sequence deduced from the
nucleotide sequence. The underlined sequence
was determined by Edman degradation.
Fig. 4. Sphingomyelin-hydrolyzing activity of wild-type and H151A
mutant SMC expressed by E. coli. The enzyme activity was measured
using an AmplexÒ Red Sphingomyelinase Assay Kit. The data plotted
represent the mean ± SEM (n ¼ 3).
Ó FEBS 2004 Insecticidal sphingomyelinase from B. cereus (Eur. J. Biochem. 271) 605
system or its nonselective damage of various organs
causes the insecticidal effect.
In conclusion, we have isolated a B. cereus strain as an
insecticidal protein producer from the larvae of M. bore.
We have found, by evaluating the insecticidal activities of
the native and recombinant proteins, that SMC produced
by B. cereus is able to kill insects rapidly when injected at
low doses. Site-directed mutagenesis revealed that the
insecticidal effect of SMC is attributable to its phospho-
lipid-hydrolyzing activity. Although the mechanism under-
lying the rapid insecticidal action remains to be resolved,
these results contribute to our understanding of the role of
the insecticidal toxin produced by B. cereus in the relation-
ship with host insects and the mechanism underlying the
insect toxicity of SMC.

Acknowledgements
This research was supported in part by the program for Basic Research
Activities for Innovative Biosciences (Bio-oriented Technology
Research Advancement Institution: BRAIN) of Japan. We thank
Professor Ryutaro Utsumi of Kinki University for his technical advice.
We are also grateful to Sumitomo Chemical Co. Ltd for supplying
cockroaches.
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