One of these active proteins was purified from the culture broth of B.cereus using anion-exchange and gel-filtration chro-matography.. A recombinant sphingomyelinase C expressed in Escheri
Trang 1Purification and functional characterization of insecticidal
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 cereusisolated 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 coliwas 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 25C 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 26C and 60% 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, after 5 lL 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: kmatsuda@nara.kindai.ac.jp
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)
Trang 2Isolation of the bacteria from antlionM 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 25C 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
25C for 16 h with shaking This preculture was added to
2 mL fresh SCD broth at a final A600value of 0.05, and then
the second culture was shaken at 25C 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 ofB 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 mM
MgSO4, 0.2 mM dNTP and 100 ng genomic DNA by
the following protocol (final volume, 50 lL): 94C for
2 min followed by 30 cycles of 94C for 15 s, 50 C for
30 s, and 68C for 2 min The amplified DNA band was
purified using low melting point agarose (Promega) and
cloned into pCRScpirtTM Amp SK(+) cloning vector
(Stratagene) The 16S rRNA gene was sequenced by the
dye-terminator method using DYEnamic ETTerminator
Cycle Sequencing Kit (Amersham Biosciences Co.,
Piscataway,
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 ofB 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 lL using a membrane with a
cut-off molecular mass of 10 kDa (Centriprep YM-10;
Millipore) Some of the solution was heated at 100C
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 mM potassium phosphate buffer (KPB,
pH 7.5) at 37C (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 byB cereus B.cereuswas 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 25C for 20 h, and an aliquot was added to
10 mL SCD broth (pH 8.0, initial A600 0.05), which was cultured at 25C for 6 h Then the broth was added to
500 mL SCD medium (pH 8.0) at a final A600value of 0.05 After the bacteria had been cultured with shaking for 8 h at
25C, the broth was centrifuged at 10 000 g for 20 min at
4C, 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 4C and dissolved in 7 mL 25 mMKPB (pH 7.5) containing 1 mM 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 mMKPB (pH 7.5) containing 1 mMdithiothreitol 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 mMKPB (pH 7.5) containing 1 mM dithiothreitol, and absorbed proteins were eluted from the resin by increasing the KCl concentration in KPB stepwise from zero to 150 mMand
500 mM 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 4C, each protein pellet was dissolved in 25 mM
KPB (pH 7.5) containing 1 mMdithiothreitol The fraction eluted with the buffer containing 150 mMKCl 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 mMKPB (pH 7.5) containing 1 mMdithiothreitol
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]
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
byB 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
Trang 3after 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 ofB 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 mM MgSO4,
0.2 mM dNTP and 100 ng genomic DNA as template
according to the following protocol: 94C for 2 min
followed by 30 cycles of 94C for 15 s, 53 C for 30 s,
and 68C for 1.5 min The amplified gene was cloned into
pCRScpirtTMAmp SK(+) cloning vector Then the cloned
gene was sequenced using the dye-terminator method with a
DYEnamic ETTerminator Cycle Sequencing Kit and
ABI3100 analyzer
Functional expression of the insecticidal toxin (SMC)
inE coli
The SMC gene amplified by PCR using the vector sense
(VS) (5¢-GGGAATTCCATATGGAAGTGTCTACAA
ATC-3¢) and vector antisense (VA) (5¢-CCGCTCG
AGCTTCATAGAAATAGTCGCCTC-3¢) primers was
cloned into NdeI and XhoI sites of pET22b(+) vector
(Novagen) The BL21[DE3]pLysS strain (Novagen) of E.colitransformed with this expression vector containing the SMC gene was incubated at 37C for 3 h, and protein expression was then induced by addition of 1 mMisopropyl thio-b-D-galactoside After incubation at 25C 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 mMKPB (pH 7.5) and applied
to a Ni/nitrilotriacetic acid affinity column (Ni-NTA His-Bind Resin; Novagen) The column was washed with
25 mMKPB (pH 7.5), and the absorbed protein was eluted with 25 mM KPB (pH 7.5) containing 400 mM imidazole The eluted sample was further purified by gel filtration using
a Superdex 75 column 10/300 (Amersham Biosciences) with
25 mMKPB (pH 7.5)
Site-directed mutagenesis The B.cereus SMC gene cloned in the pET22b expression vector was used as a template for mutagenesis T he 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 mM
MgSO4and 0.2 mMdNTP mixture in a 50-lL solution at
94C for 2 min followed by 30 cycles of 94 C for 15 s,
50C for 30 s, and 68 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 mM MgSO4 and 0.2 mM dNTP mixture in a 50-lL solution at 94C for 2 min followed by
35 cycles of 94C for 15 s, 50 C for 30 s, and 68 C for
2 min, yielding a single band of predicted size The DNA band was digested with NdeI and XhoI, and cloned into the NdeI and XhoI 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 Amplex Red Sphingomyelinase Assay Kit (Molecular Probes) For the measurement, the buffer was replaced with 100 mM
Tris/HCl buffer (pH 7.5) using a Superdex 75 column 10/
300 To 5 lL 100 mMTris/HCl buffer (pH 7.5) containing sphingomyelin (200, 500, 1000, 2000, 3000 and 4000 lM) and 2% Triton X-100 in each well of a 96-well microplate (OptiplateTM 96F; Packard Instrument Co.) was added
45 lL 100 mMTris/HCl buffer (pH 7.5) containing 10 mM
MgCl2, 100 lMAmplex red reagent, 2 UÆmL)1horseradish 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 37C, 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-tration in KPB with 1 m 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.
Trang 4intensity data were fitted usingPRISMsoftware (Graphpad
software Inc., San Diego,
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 (
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 50C, 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 100C, 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 Through
fraction, which was not absorbed by the resin, and
KCl-eluted fractions, which were KCl-eluted by increasing the KCl
concentration in the buffer (Fig 1A) The MPD value of
the Through fraction was 328 ± 194 (n¼ 2, mean ±
SEM), whereas that of the fractions eluted by 150 mMKCl
and 500 mMKCl were 167 ± 63 (n¼ 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 mMKCl-eluted fraction by gel filtration
The fraction eluted from DEAE-Sepharose by KPB
containing 150 mM 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, n¼ 2) The
34-kDa protein was able to intoxicate the cockroaches with
a MPD of 262 ± 29 ng protein/insect (mean ± SEM,
n¼ 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 T o 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 T he 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 (n¼ 3), a value close to that of the native protein, and
110 ± 10 (n¼ 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 50C 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].
Trang 5enzyme 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 (Kd 50 lM), 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 (n¼ 3); for cutworms, > 635 (n¼ 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 Amplex Red Sphingomyelinase Assay Kit The data plotted
represent the mean ± SEM (n ¼ 3).
Trang 6system 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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