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: 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) 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. 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Purification and functional characterization of insecticidal sphingomyelinase C produced by Bacillus cereus Hisashi Nishiwaki, Katsuhiko Ito, Katsuhiko Otsuki, Hiroyuki Yamamoto, Koichiro. 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 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,