- Email: [email protected]
Identification of the Functional Site in the Mosquito Larvicidal Binary Toxin of Bacillus sphaericus 1593M by Site-Directed Mutagenesis
Biochemical and Biophysical Research Communications 276, 1048 –1055 (2000) doi:10.1006/bbrc.2000.3575, available online at http://www.idealibrary.com on
Identification of the Functional Site in the Mosquito Larvicidal Binary Toxin of Bacillus sphaericus 1593M by Site-Directed Mutagenesis G. Elangovan,* M. Shanmugavelu,† F. Rajamohan,‡ D. H. Dean,§ and Kunthala Jayaraman* ,1 *Centre for Biotechnology, Anna University, Chennai, India; †Torrey Pines Institute for Molecular Studies, San Diego, California; ‡Department of Protein Engineering, Wayne Hughes Institute, Minneapolis, Minnesota; and §Department of Biochemistry, Ohio State University, Columbus, Ohio
Received August 15, 2000
To study the mode of action of the binary toxin (51and 42-kDa) of Bacillus sphaericus, amino acid residues were substituted at selected sites of the N- and C-terminal regions of both peptides. Bioassay results of the mutant binary toxins tested against mosquito larvae, Culex quinquefasciatus, revealed that most of the substitutions made on both peptides led to either decrease or total loss of the activity. Furthermore, receptor binding studies carried out for some of the mutants of the 42-kDa peptide showed mutations in N- and C-terminal regions of the 42-kDa peptide did not affect the binding of the binary toxin to brush border membrane vesicles of mosquito larvae. One of the mutants having a single amino acid substitution at the C-terminal region ( 312 R) of the 42-kDa peptide completely abolished the biological activity, implicating the role of this residue in membrane pore formation. These results indicate the importance of the C-terminal region of the 42-kDa of binary toxin, in general, and particularly the residue 312 R for biological activity against mosquito larvae. © 2000 Academic Press Key Words: Bacillus sphaericus; binary toxin; mode of action; site-directed mutagenesis; mosquito larvae; bioassay; receptor binding.
Bacillus sphaericus is a gram positive, aerobic and spore-forming organism. This bacterium synthesizes mosquito larvicidal proteins during its sporulation (51and 42-kDa) and vegetative phases (100-kDa). Among them, the 51- and 42-kDa peptides act together as a binary toxin and both the peptides are required in equal concentrations for maximum biological activity 1
To whom correspondence should be addressed. Fax: 91-442354119. E-mail: [email protected] or kayjays@giasmd01. vsnl.net.in.
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
(1–3). The binary toxin genes have been identified from a number of B. sphaericus strains, which show minor variations in the nucleotide and amino acid sequences between them (4). The 51- and 42-kDa peptides undergo proteolytic processing in the alkaline pH of mosquito larval midgut leading to their conversion into active 43and 39-kDa peptides, respectively (1, 5, 6). The essential regions of the 51- and 42-kDa peptides of different strains of B. sphaericus have been identified by gene deletion studies (7–9). Subsequently, functional domains of the binary toxin have been identified by in vivo gut binding studies using deletion derivatives from N- and C-terminal regions of 51- and 42-kDa peptides (10). In vitro binding studies have confirmed that the binding of binary toxin is mediated by a specific receptor present in the susceptible mosquito larvae (11, 12). Recently, a single 60-kDa protein anchored in the mosquito midgut membrane via a glycosyl-phosphatidylinositol (GPI) anchor has been identified as the binding protein for binary toxin (13). Previously, we had reported that alanine substitution in the selected N- and C-terminal regions of the 51- and 42-kDa peptides resulted in the loss of biological activity of binary toxin (14). Also, a nontoxic mutant restored its biological activity when mixed with another nontoxic mutant of the same peptide by functional complementation (14). In the present study, we have further extended the scanning of essential regions within the functional domain by substituting the selected residues with alanine, charged and uncharged compatible residues. The bioassay and receptor binding studies carried using purified mutant toxins showed that the C-terminal region of the 42-kDa plays a crucial role for the biological activity of binary toxin.
1048
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Bacterial strains. Escherichia coli CJ236 and E. coli MV1190 were supplied with Muta-Gene phagemid in vitro mutagenesis kit (Bio-Rad, USA). E. coli BL21 (DE3) containing the prophage carrying T7 RNA polymerase gene under the control of lacUV promoter was purchased from Invitrogen (USA). The recombinant plasmid (pSV15) is from our laboratory (15). Site-directed mutagenesis. The procedure for amino acid selection and designing the mutant primer for targeted mutagenesis were followed as described earlier (14). Site-directed mutagenesis (MutaGene phagemid in vitro mutagenesis kit, Bio-Rad, USA) was performed as detailed in the manufacturer’s manual (16). DNA sequencing was carried out by Sanger’s dideoxynucleotide termination method (17) for identification of mutant clones. Expression, purification and solubilization of the toxins. The wild type plasmid and the plasmids carrying the mutant genes were transformed into E. coli BL21 (DE3) for expression and the inclusion bodies were purified according to the published protocol (18). The final pellet was solubilized in 50 mM sodium carbonate buffer (pH 9.5). The solubilized toxins were treated with 2% (by mass) trypsin at 37°C for 3–5 h and dialyzed overnight against 50 mM sodium carbonate buffer (pH 9.5). Protein concentrations of the purified protein was estimated using Lowry’s method (19) and the purity of the sample was analyzed by 10% SDS–PAGE. Iodination of the purified toxins. Iodination of trypsin activated binary toxin was performed as described earlier (20). Twenty-five micrograms of trypsin-digested toxin was labeled using 1 mCi of 125I and one IODOBEAD (Pierce), as detailed in the manufacturer’s manual. Free Iodine was separated from the toxin by passing the sample through a 2.0 ml prepacked Excellulose GF-5 column (Pierce). The specific activity of the labeled toxins was calculated. Preparation of brush border membrane vesicles (BBMV). BBMV from C. quinquefasciatus mosquito larvae were prepared by differential magnesium precipitation method as modified by Wolfersberger et al. (21). The final vesicles were resuspended in binding buffer (8 mM NaHPO 4, 2 mM KH 2PO 4, 150 mM NaCl, pH 7.4, containing 0.1% bovine serum albumin) and stored at ⫺70°C until use. Bioassay against mosquito larvae. The purified wild type and mutant binary toxins were used to carry out bioassay against second instar C. quinquefasciatus mosquito larvae. Bioassays were performed according to the WHO protocol (14, 22). Binding assays. Saturation, competition (homologous and heterologous) and dissociation binding assays were performed as per the method of Rajamohan et al. (20). After incubation at room temperature for a specific time, the samples were centrifuged at 15,500 rpm and the radioactivity in the pellet was measured (20). All binding assays were carried out in triplicates.
RESULTS AND DISCUSSION Site-Directed Mutagenesis Understanding the mechanism of action of insect larvicidal toxins is a prerequisite not only for studying the mechanism of resistance development but also to improve the potency of the toxins against susceptible insects (6). A model for the mode of action of the binary toxin of B. sphaericus has been proposed based on studies carried out using wild type and deletion derivatives of this toxin (10). This model states that the
N-terminal region of the 51-kDa peptide is responsible for larval gut binding, whereas, its C-terminal region is involved in interaction with the N-terminal region of 42-kDa, which results in the internalization of the toxin complex (10). They have also suggested that the C-terminal region of 42-kDa may be important for internalization (pore formation) of the toxin complex. In our earlier study, we had chosen alanine residue for substitution in selected sites of N- and C-terminal region of 51- and 42-kDa peptides by site-directed mutagenesis (14). In the present study, in addition to benign alanine substitution, an attempt has been made to study the effect of charged and uncharged compatible amino acids substitution in the different functional domains of the binary toxin. The location of the replaced amino acids in the mutant binary toxins and their biological activity is shown in Table 1a. The schematic explanation in Fig. 1 also shows the location of amino acids substituted for all the mutants generated and their biological activity towards mosquito larvae. Structural Stability of the Binary Toxin after Mutation The SDS–PAGE analysis of mutant binary toxins showed that there is no difference either in the expression levels or structure of mutants compared to wild type toxin (Fig. 2a). However, after trypsin digestion, some of the 42 N-terminal mutants (42N3, 42N4, 42N5 & 42N6) showed degradation in the 42-kDa peptide (having intact 51-kDa peptide) in the SDS–PAGE. This could be due to the exposure of the tryptic cleavage site ( 49R), present in the 42 N-terminal region (Fig. 2b). Mutations at the N- and C-Terminal Regions of the 51-kDa Peptide The bioassay results of mutant toxins against mosquito larvae revealed that alanine substitution in two of the 51 N-terminal regions; viz. 16KK 17 3 AA (51N1) and 16KKF 18 3 AAA (51N2) did not affect the biological activity. This reconfirms the earlier observation on the nonessentiality of this region, as it is located upstream to the proteolytic cleavage sites of the 51-kDa peptide (8, 10). Replacement of three amino acids in the N-terminal regions, 32YNL 34 3 AAA (51N3) and 38 SKK 40 3 AAA (51N4) by alanine blocks resulted in total loss of biological activity. The secondary structure prediction of 51-kDa peptide shows that the amino acids 1–34 are potential loop forming regions, followed by ␣-helical structure up to 45 amino acids. Thus, either a direct disturbance of the loop structure (51N3) or ␣-helix (51N4) in this region may affect the gut (receptor) binding activity of the toxin. Earlier studies have shown that the deletion of 41 amino acids from the N-terminal region of the 51-kDa peptide of B. sphaericus 2362 destroyed its biological activity (8).
1049
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1a
Biological Activity of the Mutant Binary Toxins against C. quinquefasciatus Mosquito Larvae Mutated site in the binary toxin Mutant name
Region of mutation
1. pSV15 2. 51N1 3. 51N2 4. 51N3 5. 51C1 6. 51C3 7. 42N3 8. 42N4 9. 42N5 10. 42N6 11. 42C1 12. 42C3 13. 42C4 14. 42C5 15. 42C6 16. 42C7
Wild type 51-N 51-N 51-N 51-C 51-C 42-N 42-N 42-N 42-N 42-C 42-C 42-C 42-C 42-C 42-C
51-kDa N________C _2______ _2______ __2_____ _____2__ ______2_ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________
42-kDa N________C ________ ________ ________ ________ ________ __2_____ __2_____ __2_____ __2_____ _____2__ ______2_ ______2_ ______2_ ______2_ ______2_
Amino acid(s) substituted
LC 50 in 24 hours (mean and SD)
Toxicity
— 16 KK 17/ 16AA 17 16 KKF 18/ 16AAA 18 32 YNL 34/ 32AAA 34 387 YRL 389/ 387AAA 389 408 KH 409/ 408AA 409 N 52H N 52A Q 53M Q 53A 294 LLI 296/ 294AAA 296 326 HR 327/ 326AA 327 R 312K R 312H R 312I E 303A
20.7 ⫾ 2.05 ng 21.0 ⫾ 2.16 ng 21.3 ⫾ 2.86 ng * * 203.3 ⫾ 26.2 ng 126.6 ⫾ 12.5 ng 105.0 ⫾ 10.8 ng 95.0 ⫾ 8.2 ng 83.3 ⫾ 6.2 ng 2.06 ⫾ 0.25 g * * * * *
Toxic Toxic Toxic Nontoxic Nontoxic Toxic Toxic Toxic Toxic Toxic Less toxic Nontoxic Nontoxic Nontoxic Nontoxic Nontoxic
Note. LC 50 values were expressed in terms of protein concentration/ml (average of triplicates). SD, standard deviations. * No mortality of larvae was observed with up to 10 g/ml protein concentration (i.e., nontoxic).
Mutations in the C-terminal region of the 51-kDa peptide resulted in tenfold reduction in toxicity ( 408KH 409 3 AA, 51C3). Total loss of activity was observed with mutants 51C1 ( 387YRL 389 3 AAA) and 51C2 ( 392IQ 393 3 AA) (Table 1a). These results are in agreement with the previous results of deletion studies showing that the C-terminal region of the 51-kDa peptide is involved in the interaction with N-terminal region of 42-kDa peptide (8). In vitro binding studies using these toxins are in progress to identify the residues involved in the receptor binding. Mutations at the N- and C-Terminal Regions of the 42-kDa Peptide Single alanine substitution in the N-terminal region of the 42-kDa peptide ( 47C 3 A, 42N2) also led to total loss of biological activity against mosquito larvae. The other two single amino acid substitution at the 42 N-terminal regions ( 52N 3 A; 52N 3 H and 53Q 3 A; 53 Q 3 M) retained the biological activity, albeit four- to sixfold lower (Table 1a). This indicates that the residues 52N and 53Q may not play a crucial role for biological activity. These results are in agreement with the proposed interactions between the C- and N-terminal regions of 51- and 42-kDa peptides, respectively, to form the functional binary toxin. The mutation made in the C-terminal region of the 42-kDa peptide ( 293LLI 2953 AAA, 42C1) resulted in a 100-fold reduction in toxicity. Other mutations, 303E 3 A (42C7), 312R 3 A, K, H & I (42C2, 42C4, 42C5, and
42C6) and 325HR 326 3 AA (42C3) lost their total biological activity. Previously, it has been reported that deletion of more than 17 amino acids from the C-terminal region of this peptide resulted in loss of toxicity (7, 9, 10). Substitution of C-terminal arginine ( 312R) with alanine as well as other compatible charged amino acids (lysine and histidine) or an uncharged amino acid (isoleucine) completely abolished the biological activity of the toxin. Glutamate ( 303E) when substituted with alanine also completely abolished the activity of the toxin (Table 1a). These results suggest that these two residues ( 312R and 303E) either individually or in combination may play an important role in pore formation and subsequent internalization of toxin complexes. Functional Complementation between Two Nontoxic Mutant Toxins Previously, we had reported that when two nontoxic mutants of either the 51- or the 42-kDa peptide were mixed and tested for toxicity, biological activity was restored by functional complementation (14). For example, a mixture of the two nontoxic mutant binary toxins, 51N4 ⫹ 51C1 (retaining the 42-kDa intact) and 42N2 ⫹ 42C2 (retaining the 51-kDa intact) were toxic to mosquito larvae. Functional complementation between two nontoxic mutants suggests that oligomerisation of the ingested subunits takes place in the midgut of the mosquito larvae. We have further extended our complementation studies with all the nontoxic mutants reported in this study (Table 1b). These results
1050
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1b
Regaining the Biological Activity of Some of the Nontoxic Mutant Binary Toxins by Functional Complementation (upon Mixing Two Nontoxic Mutant Toxins) Mutated site in the binary toxin Mutants Combinations 1. 51N4 } ⫹ 51C1 2. 51C1 ⫹ 51C2 } 3. 42N2 } ⫹ 42C3 }
4. 42C2 ⫹ 42C3 5. 42C4 ⫹ 42C5 6. 42C4 ⫹ 42C6 7. 42C4 ⫹ 42C7 8. 42C5 ⫹ 42C6 9. 42C5 ⫹ 42C7 10. 42C6 ⫹ 42C7
51-kDa
42-kDa
N_2______C ______2_ _____2__ ______2_ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________
N________C ________ ________ ________ _2______ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_
LC 50 in 24 hours (mean and SD)
Toxicity
423.3 ⫾ 20.5 ng
Toxic
* 406.6 ⫾ 24.6 ng
Nontoxic Toxic
*
Nontoxic
*
Nontoxic
*
Nontoxic
506.6 ⫾ 24.9 ng *
Toxic Nontoxic
636.6 ⫾ 49.9 ng
Toxic
523.3 ⫾ 37.9 ng
Toxic
Note. LC 50 values were expressed in terms of protein concentration/ml (average of triplicates). SD, standard deviations. * No mortality of larvae was observed with up to 10 g/ml protein concentration (i.e., nontoxic). }Mutants already published (Shanmugavelu et al., 1998a), which were used for complementation assays in the present study.
suggest that a nontoxic mutant having mutation at one site can be complemented by a second nontoxic mutant peptide with a mutation at a different site. For example, 42C7 could be complemented by the nontoxic mutants, 42C4, 42C5, and 42C6, though all these individual mutations are at the critical 303E and 312R residues. When the mutant 42C7 is mixed with other mutants they may form oligomers and result in the availability of 303E and 312R residues that are crucial for biological activity. In contrast, the mixtures of 42C4 ⫹ 42C5, 42C4 ⫹ 42C6 and 42C5 ⫹ 42C6 remained nontoxic. This may be due to the fact that the arginine residue ( 312R) is mutated in all these three mutants, as a result a crucial arginine residue is not available at this position. A similar result was obtained with the mixtures of 51C1 ⫹ 51C2 and 42C2 ⫹ 42C3, implying that two different nontoxic mutant peptides having sites of substitutions located close to each other thereby could not functionally complement each other (Table 1b). BINDING STUDIES It was reported that the 51-kDa peptide of the binary toxin is involved in the gut (receptor) binding (10, 13). It has also been reported that both 51- and 42-kDa peptides bind to the gastric caecae and posterior midgut of Culex larvae when administered together (23).
Hence, it was of interest to determine whether mutations at the 42-kDa peptide have any role on the binding of binary toxin to BBMV. To understand this, binding patterns of both the wild type and some of the 42-kDa mutants (N 52H, N 52A, Q 53M, Q 53A, R 312K, and R 312H), with intact 51-kDa were studied using mosquito BBMV. Saturation binding, competition binding (homologous and heterologous) and dissociation kinetics studies were carried out using the solubilized, purified, 125I-labeled wild type and mutant binary toxins. All bioassays were carried out in triplicates. Saturation Binding Assay Saturation binding assays were performed by incubating with increasing concentrations (1 to 3 nM) of labeled toxin with fixed quantities (200 g/ml) of BBMV. After 1 h incubation at room temperature, the samples were collected and the amount of toxin bound to the BBMV was calculated by measuring the radioactivity in the pellet (20). The amount of toxin required to achieve saturation binding was similar for both wild type and mutant binary toxins (Fig. 3a). This is in accordance with the earlier reports that all mutants of 42 N-terminal region, which is involved in interaction with the 51-kDa peptide, but not with the receptor, retained toxicity. Whereas the 42
1051
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Line diagram showing the mutations generated at different sites of 51- and 42-kDa peptides of the binary toxin of B. sphaericus by site-directed mutagenesis and their biological activity towards C. quinquefasciatus mosquito larvae. (⫺), Nontoxic up to 10 g/ml protein concentration. (⫹), Toxic.
C-terminal domain (nontoxic mutants 42C4 and 42C5) is not involved in this interaction, lost its biological activity, because of the substitution of crucial amino acids in this region.
recognize the same binding site(s) as that of the wild type toxin. When labeled wild type toxin was competed with excess unlabeled mutant toxins, all the mutants competed as efficiently for the wild type binding site(s) as did unlabeled wild type binary toxin (Fig. 3c).
Competition Membrane Binding Assay In homologous competition binding assays, binding of the labeled toxins was competed with an excess concentration of the corresponding unlabeled toxin to evaluate the binding affinity and affinity concentrations on mosquito larval BBMV. This assay can distinguish between reversible and irreversible binding. Our results showed that the wild type and the mutant binary toxins bind to the BBMV with similar binding affinity (Fig. 3b). Both the wild type and mutant binary toxins bind to the BBMV in a reversible manner. This is in contrast to the binding of Cry toxins of B. thuringiensis, where the binding is mostly irreversible (24). Heterologous competition experiments were performed to evaluate whether the mutant binary toxins
Dissociation Binding Assay Since all the toxins (wild type and mutants) showed similar binding affinities to BBMV, we tested the dissociation kinetics of the BBMV-bound toxins. The labeled toxins were first allowed to bind to the BBMV and were then chased with the corresponding unlabeled toxins. Our results showed that 60 to 80% of the BBMV-bound wild type and mutant toxins were displaced by the addition of excess concentration of unlabeled ligands (i.e., both wild type and mutant binary toxins were reversibly associated), which is shown in the graph (Fig. 3d). The binding assays of wild type and 42-kDa mutant binary toxins against mosquito BBMV show that bind-
1052
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. (a) SDS–PAGE profile of wild type and mutant binary toxins of B. sphaericus 1593M, solubilized in 50 mM sodium carbonate buffer, pH 9. 5. Lanes: 1, molecular weight marker; 3, wild type (pSV15); 4, 42N3; 5, 42N4; 6, 42N5; 7, 42N6; 8, 42C4; 10, 42C5; and 2 & 9, trypsin digested wild type sample. Twenty micrograms of solubilized samples were loaded in each well on a 10% SDS–polyacrylamide gel. (b) SDS–PAGE profile of Trypsin (2% w/w) digested wild type and mutant binary toxins of B. sphaericus 1593M. Lanes: 1, molecular weight marker; 9, wild type toxin (pSV15); and 2, 3, 4, 5, 6, 7, 8, & 10, Mutant toxins (42C6, 42C7, 42N3, 42N4, 42N5, 42N6, 42C4, and 42C5, respectively). Ten micrograms of purified, trypsin digested samples were loaded in each well on a 10% SDS–polyacrylamide gel.
ing is not affected by mutations at the 42-kDa peptide. This result matches with the earlier reports showing the 51-kDa peptide of the binary toxin is involved in binding to the gut receptor (13). However, the biological activity of the binary toxin is decreased or abolished by certain mutations, which were made in the 42-kDa peptide. For example, mutation of 312R residue in the 42 C-terminal completely destroys the biological activity of the binary toxin, even though there is no difference in the binding pattern of this mutant towards mosquito BBMV. The loss of activity of binary toxin
when the residue 312R of 42-kDa was substituted with other positively charged amino acids, viz. lysine and histidine points to the central significance of the arginyl residue possibly in membrane pore formation. This result is in agreement with the previous reports that the C-terminal region of the 42-kDa may be important for pore formation of the toxin. In conclusion, we have identified certain amino acid residues at the N- and C-terminal regions of 51- and 42-kDa peptides that are crucial for the biological activity of the binary toxin. In vitro binding assays with
1053
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. (a) Saturation binding of wild type (pSV15) and mutant (42N3, 42N4, 42N5, 42N6, 42C4, and 42C5) binary toxins of B. sphaericus with C. quinquefasciatus BBMV. (b) Homologous competition binding of wild type (pSV15) and mutant (42N3, 42N4, 42N5, 42N6, 42C4, and 42C5) binary toxins of B. sphaericus with C. quinquefasciatus BBMV. (c) Heterologous competition binding of wild type (pSV15) binary toxin by mutant (42N3, 42N4, 42N5, 42N6, 42C4, and 42C5) toxins of B. sphaericus with C. quinquefasciatus BBMV.* 125I-labeled toxins. (d) Dissociation kinetics of wild type (pSV15) and mutant (42N3, 42N4, 42N5, 42N6, 42C4, and 42C5) binary toxins of B. sphaericus with C. quinquefasciatus BBMV.
1054
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
other mutants of 51 N- and C-terminal region will further enable us in understanding the mode of action of this toxin. Further, the role of residues 327R and 303E towards biological activity and binding to BBMV is under investigation. ACKNOWLEDGMENTS We thank April Curtiss of the Department of Biochemistry, Ohio State University, Columbus, Ohio, and Dr. Geetha of Centre for Biotechnology, Anna University, Chennai, India, for their help and discussion of this work. We acknowledge Indo-Swiss Collaboration in Biotechnology, Switzerland, Council of Scientific and Industrial Research, New Delhi, India, and Department of Biotechnology, Government of India, for financial support of the project.
REFERENCES 1. Baumann, P., Clark, M. A., Baumann, L., and Broadwell, A. H. (1991) Microbiol. Rev. 55, 425– 436. 2. Porter, A. G., Davidson, E. W., and Liu, J. W. (1993) Microbiol. Rev. 57, 838 – 861. 3. Shanmugavelu, M., Elangovan, G., Kathirvel, M., and Jayaraman, K. (1998b) Biotech. Lett. 20(4), 341–344. 4. Berry, C., Jackson-Yap, J., Oei, C., and Hindley, J. (1989) Nucleic Acids Res. 17, 7516. 5. Davidson, E. W. (1988) J. Med. Entomol. 25, 151–157. 6. Charles, J. -F., Nielson-LeRoux, C., and Delecluse, A. (1996) Ann. Rev. Entomol. 42, 451– 472. 7. Broadwell, A. H., Clark, M. A., Baumann, L., and Baumann, P. (1990) J. Bacteriol. 172, 4032– 4036. 8. Clark, M. A., and Baumann, P. (1990). J. Bacteriol. 172, 6759 – 6763.
9. Sebo, P., Bennardo, D., De la Torre, F., and Szulmajster, J. (1990) Eur. J. Biochem. 194, 161–165. 10. Oei, C., Hindley, J., and Berry, C. (1992) J. Gen. Microbiol. 138, 1515–1526. 11. Nielsen-LeRoux, C., and Charles, J. F. (1992) Eur. J. Biochem. 210, 585–590. 12. Silva-Filha, M. H., Nielsen-LeRoux, C., and Charles, J. -F. (1997) Eur. J. Biochem. 210, 585–590. 13. Silva-Filha, M. H., Nielsen-LeRoux, C., and Charles, J. -F. (1999) Insect Biochem. Mol. Biol. 29, 711–721. 14. Shanmugavelu, M., Rajamohan, F., Kathirvel, M., Elangovan, G., Dean, D. H., and Jayaraman, K. (1998a) Appl. Environ. Microbiol. 64, 756 –759. 15. Shanmugavelu, M., Meenakshisundaram, B., and Jayaraman, K. (1997) Biotechnol. Lett. 19, 341–344. 16. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488 – 492. 17. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463–5467. 18. Almond, B. D., and Dean, D. H. (1993) Appl. Environ. Microbiol. 59, 2442–2448. 19. Lowry, O. H., Rosebrough, N. H., Farr, N. J., and Randal, R. J. (1951) J. Biol. Chem. 193, 265–268. 20. Rajamohan, F., Alcantara, E., Lee, M. K., Chen, X. J., Curtiss, A., and Dean, D. H. (1995) J. Bacteriol. 177, 2276 –2282. 21. Wolfersberger, M. G., Luthy, P., Maurer, A., Parenti, P., Sacchi, F. V., Gioordana, B., and Hanozet, G. M. (1987) Comp. Biochem. Biophysiol. 86, 301–308. 22. de Barjac, H. (1990) Rutgers University Press, Brunswick, N J. 23. Davidson, E. W., Oei, C., Meyer, M., Beiber, A. L., Hindley, J., and Berry, C. (1990) Can. J. Microbiol. 36, 870 – 878. 24. Liang, Y., Patel, S. S., and Dean, D. H. (1995) J. Biol. Chem. 270, 24719 –24724.
1055
Identification of the Functional Site in the Mosquito Larvicidal Binary Toxin of Bacillus sphaericus 1593M by Site-Directed Mutagenesis G. Elangovan,* M. Shanmugavelu,† F. Rajamohan,‡ D. H. Dean,§ and Kunthala Jayaraman* ,1 *Centre for Biotechnology, Anna University, Chennai, India; †Torrey Pines Institute for Molecular Studies, San Diego, California; ‡Department of Protein Engineering, Wayne Hughes Institute, Minneapolis, Minnesota; and §Department of Biochemistry, Ohio State University, Columbus, Ohio
Received August 15, 2000
To study the mode of action of the binary toxin (51and 42-kDa) of Bacillus sphaericus, amino acid residues were substituted at selected sites of the N- and C-terminal regions of both peptides. Bioassay results of the mutant binary toxins tested against mosquito larvae, Culex quinquefasciatus, revealed that most of the substitutions made on both peptides led to either decrease or total loss of the activity. Furthermore, receptor binding studies carried out for some of the mutants of the 42-kDa peptide showed mutations in N- and C-terminal regions of the 42-kDa peptide did not affect the binding of the binary toxin to brush border membrane vesicles of mosquito larvae. One of the mutants having a single amino acid substitution at the C-terminal region ( 312 R) of the 42-kDa peptide completely abolished the biological activity, implicating the role of this residue in membrane pore formation. These results indicate the importance of the C-terminal region of the 42-kDa of binary toxin, in general, and particularly the residue 312 R for biological activity against mosquito larvae. © 2000 Academic Press Key Words: Bacillus sphaericus; binary toxin; mode of action; site-directed mutagenesis; mosquito larvae; bioassay; receptor binding.
Bacillus sphaericus is a gram positive, aerobic and spore-forming organism. This bacterium synthesizes mosquito larvicidal proteins during its sporulation (51and 42-kDa) and vegetative phases (100-kDa). Among them, the 51- and 42-kDa peptides act together as a binary toxin and both the peptides are required in equal concentrations for maximum biological activity 1
To whom correspondence should be addressed. Fax: 91-442354119. E-mail: [email protected] or kayjays@giasmd01. vsnl.net.in.
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
(1–3). The binary toxin genes have been identified from a number of B. sphaericus strains, which show minor variations in the nucleotide and amino acid sequences between them (4). The 51- and 42-kDa peptides undergo proteolytic processing in the alkaline pH of mosquito larval midgut leading to their conversion into active 43and 39-kDa peptides, respectively (1, 5, 6). The essential regions of the 51- and 42-kDa peptides of different strains of B. sphaericus have been identified by gene deletion studies (7–9). Subsequently, functional domains of the binary toxin have been identified by in vivo gut binding studies using deletion derivatives from N- and C-terminal regions of 51- and 42-kDa peptides (10). In vitro binding studies have confirmed that the binding of binary toxin is mediated by a specific receptor present in the susceptible mosquito larvae (11, 12). Recently, a single 60-kDa protein anchored in the mosquito midgut membrane via a glycosyl-phosphatidylinositol (GPI) anchor has been identified as the binding protein for binary toxin (13). Previously, we had reported that alanine substitution in the selected N- and C-terminal regions of the 51- and 42-kDa peptides resulted in the loss of biological activity of binary toxin (14). Also, a nontoxic mutant restored its biological activity when mixed with another nontoxic mutant of the same peptide by functional complementation (14). In the present study, we have further extended the scanning of essential regions within the functional domain by substituting the selected residues with alanine, charged and uncharged compatible residues. The bioassay and receptor binding studies carried using purified mutant toxins showed that the C-terminal region of the 42-kDa plays a crucial role for the biological activity of binary toxin.
1048
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Bacterial strains. Escherichia coli CJ236 and E. coli MV1190 were supplied with Muta-Gene phagemid in vitro mutagenesis kit (Bio-Rad, USA). E. coli BL21 (DE3) containing the prophage carrying T7 RNA polymerase gene under the control of lacUV promoter was purchased from Invitrogen (USA). The recombinant plasmid (pSV15) is from our laboratory (15). Site-directed mutagenesis. The procedure for amino acid selection and designing the mutant primer for targeted mutagenesis were followed as described earlier (14). Site-directed mutagenesis (MutaGene phagemid in vitro mutagenesis kit, Bio-Rad, USA) was performed as detailed in the manufacturer’s manual (16). DNA sequencing was carried out by Sanger’s dideoxynucleotide termination method (17) for identification of mutant clones. Expression, purification and solubilization of the toxins. The wild type plasmid and the plasmids carrying the mutant genes were transformed into E. coli BL21 (DE3) for expression and the inclusion bodies were purified according to the published protocol (18). The final pellet was solubilized in 50 mM sodium carbonate buffer (pH 9.5). The solubilized toxins were treated with 2% (by mass) trypsin at 37°C for 3–5 h and dialyzed overnight against 50 mM sodium carbonate buffer (pH 9.5). Protein concentrations of the purified protein was estimated using Lowry’s method (19) and the purity of the sample was analyzed by 10% SDS–PAGE. Iodination of the purified toxins. Iodination of trypsin activated binary toxin was performed as described earlier (20). Twenty-five micrograms of trypsin-digested toxin was labeled using 1 mCi of 125I and one IODOBEAD (Pierce), as detailed in the manufacturer’s manual. Free Iodine was separated from the toxin by passing the sample through a 2.0 ml prepacked Excellulose GF-5 column (Pierce). The specific activity of the labeled toxins was calculated. Preparation of brush border membrane vesicles (BBMV). BBMV from C. quinquefasciatus mosquito larvae were prepared by differential magnesium precipitation method as modified by Wolfersberger et al. (21). The final vesicles were resuspended in binding buffer (8 mM NaHPO 4, 2 mM KH 2PO 4, 150 mM NaCl, pH 7.4, containing 0.1% bovine serum albumin) and stored at ⫺70°C until use. Bioassay against mosquito larvae. The purified wild type and mutant binary toxins were used to carry out bioassay against second instar C. quinquefasciatus mosquito larvae. Bioassays were performed according to the WHO protocol (14, 22). Binding assays. Saturation, competition (homologous and heterologous) and dissociation binding assays were performed as per the method of Rajamohan et al. (20). After incubation at room temperature for a specific time, the samples were centrifuged at 15,500 rpm and the radioactivity in the pellet was measured (20). All binding assays were carried out in triplicates.
RESULTS AND DISCUSSION Site-Directed Mutagenesis Understanding the mechanism of action of insect larvicidal toxins is a prerequisite not only for studying the mechanism of resistance development but also to improve the potency of the toxins against susceptible insects (6). A model for the mode of action of the binary toxin of B. sphaericus has been proposed based on studies carried out using wild type and deletion derivatives of this toxin (10). This model states that the
N-terminal region of the 51-kDa peptide is responsible for larval gut binding, whereas, its C-terminal region is involved in interaction with the N-terminal region of 42-kDa, which results in the internalization of the toxin complex (10). They have also suggested that the C-terminal region of 42-kDa may be important for internalization (pore formation) of the toxin complex. In our earlier study, we had chosen alanine residue for substitution in selected sites of N- and C-terminal region of 51- and 42-kDa peptides by site-directed mutagenesis (14). In the present study, in addition to benign alanine substitution, an attempt has been made to study the effect of charged and uncharged compatible amino acids substitution in the different functional domains of the binary toxin. The location of the replaced amino acids in the mutant binary toxins and their biological activity is shown in Table 1a. The schematic explanation in Fig. 1 also shows the location of amino acids substituted for all the mutants generated and their biological activity towards mosquito larvae. Structural Stability of the Binary Toxin after Mutation The SDS–PAGE analysis of mutant binary toxins showed that there is no difference either in the expression levels or structure of mutants compared to wild type toxin (Fig. 2a). However, after trypsin digestion, some of the 42 N-terminal mutants (42N3, 42N4, 42N5 & 42N6) showed degradation in the 42-kDa peptide (having intact 51-kDa peptide) in the SDS–PAGE. This could be due to the exposure of the tryptic cleavage site ( 49R), present in the 42 N-terminal region (Fig. 2b). Mutations at the N- and C-Terminal Regions of the 51-kDa Peptide The bioassay results of mutant toxins against mosquito larvae revealed that alanine substitution in two of the 51 N-terminal regions; viz. 16KK 17 3 AA (51N1) and 16KKF 18 3 AAA (51N2) did not affect the biological activity. This reconfirms the earlier observation on the nonessentiality of this region, as it is located upstream to the proteolytic cleavage sites of the 51-kDa peptide (8, 10). Replacement of three amino acids in the N-terminal regions, 32YNL 34 3 AAA (51N3) and 38 SKK 40 3 AAA (51N4) by alanine blocks resulted in total loss of biological activity. The secondary structure prediction of 51-kDa peptide shows that the amino acids 1–34 are potential loop forming regions, followed by ␣-helical structure up to 45 amino acids. Thus, either a direct disturbance of the loop structure (51N3) or ␣-helix (51N4) in this region may affect the gut (receptor) binding activity of the toxin. Earlier studies have shown that the deletion of 41 amino acids from the N-terminal region of the 51-kDa peptide of B. sphaericus 2362 destroyed its biological activity (8).
1049
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1a
Biological Activity of the Mutant Binary Toxins against C. quinquefasciatus Mosquito Larvae Mutated site in the binary toxin Mutant name
Region of mutation
1. pSV15 2. 51N1 3. 51N2 4. 51N3 5. 51C1 6. 51C3 7. 42N3 8. 42N4 9. 42N5 10. 42N6 11. 42C1 12. 42C3 13. 42C4 14. 42C5 15. 42C6 16. 42C7
Wild type 51-N 51-N 51-N 51-C 51-C 42-N 42-N 42-N 42-N 42-C 42-C 42-C 42-C 42-C 42-C
51-kDa N________C _2______ _2______ __2_____ _____2__ ______2_ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________
42-kDa N________C ________ ________ ________ ________ ________ __2_____ __2_____ __2_____ __2_____ _____2__ ______2_ ______2_ ______2_ ______2_ ______2_
Amino acid(s) substituted
LC 50 in 24 hours (mean and SD)
Toxicity
— 16 KK 17/ 16AA 17 16 KKF 18/ 16AAA 18 32 YNL 34/ 32AAA 34 387 YRL 389/ 387AAA 389 408 KH 409/ 408AA 409 N 52H N 52A Q 53M Q 53A 294 LLI 296/ 294AAA 296 326 HR 327/ 326AA 327 R 312K R 312H R 312I E 303A
20.7 ⫾ 2.05 ng 21.0 ⫾ 2.16 ng 21.3 ⫾ 2.86 ng * * 203.3 ⫾ 26.2 ng 126.6 ⫾ 12.5 ng 105.0 ⫾ 10.8 ng 95.0 ⫾ 8.2 ng 83.3 ⫾ 6.2 ng 2.06 ⫾ 0.25 g * * * * *
Toxic Toxic Toxic Nontoxic Nontoxic Toxic Toxic Toxic Toxic Toxic Less toxic Nontoxic Nontoxic Nontoxic Nontoxic Nontoxic
Note. LC 50 values were expressed in terms of protein concentration/ml (average of triplicates). SD, standard deviations. * No mortality of larvae was observed with up to 10 g/ml protein concentration (i.e., nontoxic).
Mutations in the C-terminal region of the 51-kDa peptide resulted in tenfold reduction in toxicity ( 408KH 409 3 AA, 51C3). Total loss of activity was observed with mutants 51C1 ( 387YRL 389 3 AAA) and 51C2 ( 392IQ 393 3 AA) (Table 1a). These results are in agreement with the previous results of deletion studies showing that the C-terminal region of the 51-kDa peptide is involved in the interaction with N-terminal region of 42-kDa peptide (8). In vitro binding studies using these toxins are in progress to identify the residues involved in the receptor binding. Mutations at the N- and C-Terminal Regions of the 42-kDa Peptide Single alanine substitution in the N-terminal region of the 42-kDa peptide ( 47C 3 A, 42N2) also led to total loss of biological activity against mosquito larvae. The other two single amino acid substitution at the 42 N-terminal regions ( 52N 3 A; 52N 3 H and 53Q 3 A; 53 Q 3 M) retained the biological activity, albeit four- to sixfold lower (Table 1a). This indicates that the residues 52N and 53Q may not play a crucial role for biological activity. These results are in agreement with the proposed interactions between the C- and N-terminal regions of 51- and 42-kDa peptides, respectively, to form the functional binary toxin. The mutation made in the C-terminal region of the 42-kDa peptide ( 293LLI 2953 AAA, 42C1) resulted in a 100-fold reduction in toxicity. Other mutations, 303E 3 A (42C7), 312R 3 A, K, H & I (42C2, 42C4, 42C5, and
42C6) and 325HR 326 3 AA (42C3) lost their total biological activity. Previously, it has been reported that deletion of more than 17 amino acids from the C-terminal region of this peptide resulted in loss of toxicity (7, 9, 10). Substitution of C-terminal arginine ( 312R) with alanine as well as other compatible charged amino acids (lysine and histidine) or an uncharged amino acid (isoleucine) completely abolished the biological activity of the toxin. Glutamate ( 303E) when substituted with alanine also completely abolished the activity of the toxin (Table 1a). These results suggest that these two residues ( 312R and 303E) either individually or in combination may play an important role in pore formation and subsequent internalization of toxin complexes. Functional Complementation between Two Nontoxic Mutant Toxins Previously, we had reported that when two nontoxic mutants of either the 51- or the 42-kDa peptide were mixed and tested for toxicity, biological activity was restored by functional complementation (14). For example, a mixture of the two nontoxic mutant binary toxins, 51N4 ⫹ 51C1 (retaining the 42-kDa intact) and 42N2 ⫹ 42C2 (retaining the 51-kDa intact) were toxic to mosquito larvae. Functional complementation between two nontoxic mutants suggests that oligomerisation of the ingested subunits takes place in the midgut of the mosquito larvae. We have further extended our complementation studies with all the nontoxic mutants reported in this study (Table 1b). These results
1050
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1b
Regaining the Biological Activity of Some of the Nontoxic Mutant Binary Toxins by Functional Complementation (upon Mixing Two Nontoxic Mutant Toxins) Mutated site in the binary toxin Mutants Combinations 1. 51N4 } ⫹ 51C1 2. 51C1 ⫹ 51C2 } 3. 42N2 } ⫹ 42C3 }
4. 42C2 ⫹ 42C3 5. 42C4 ⫹ 42C5 6. 42C4 ⫹ 42C6 7. 42C4 ⫹ 42C7 8. 42C5 ⫹ 42C6 9. 42C5 ⫹ 42C7 10. 42C6 ⫹ 42C7
51-kDa
42-kDa
N_2______C ______2_ _____2__ ______2_ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________ ________
N________C ________ ________ ________ _2______ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_ ______2_
LC 50 in 24 hours (mean and SD)
Toxicity
423.3 ⫾ 20.5 ng
Toxic
* 406.6 ⫾ 24.6 ng
Nontoxic Toxic
*
Nontoxic
*
Nontoxic
*
Nontoxic
506.6 ⫾ 24.9 ng *
Toxic Nontoxic
636.6 ⫾ 49.9 ng
Toxic
523.3 ⫾ 37.9 ng
Toxic
Note. LC 50 values were expressed in terms of protein concentration/ml (average of triplicates). SD, standard deviations. * No mortality of larvae was observed with up to 10 g/ml protein concentration (i.e., nontoxic). }Mutants already published (Shanmugavelu et al., 1998a), which were used for complementation assays in the present study.
suggest that a nontoxic mutant having mutation at one site can be complemented by a second nontoxic mutant peptide with a mutation at a different site. For example, 42C7 could be complemented by the nontoxic mutants, 42C4, 42C5, and 42C6, though all these individual mutations are at the critical 303E and 312R residues. When the mutant 42C7 is mixed with other mutants they may form oligomers and result in the availability of 303E and 312R residues that are crucial for biological activity. In contrast, the mixtures of 42C4 ⫹ 42C5, 42C4 ⫹ 42C6 and 42C5 ⫹ 42C6 remained nontoxic. This may be due to the fact that the arginine residue ( 312R) is mutated in all these three mutants, as a result a crucial arginine residue is not available at this position. A similar result was obtained with the mixtures of 51C1 ⫹ 51C2 and 42C2 ⫹ 42C3, implying that two different nontoxic mutant peptides having sites of substitutions located close to each other thereby could not functionally complement each other (Table 1b). BINDING STUDIES It was reported that the 51-kDa peptide of the binary toxin is involved in the gut (receptor) binding (10, 13). It has also been reported that both 51- and 42-kDa peptides bind to the gastric caecae and posterior midgut of Culex larvae when administered together (23).
Hence, it was of interest to determine whether mutations at the 42-kDa peptide have any role on the binding of binary toxin to BBMV. To understand this, binding patterns of both the wild type and some of the 42-kDa mutants (N 52H, N 52A, Q 53M, Q 53A, R 312K, and R 312H), with intact 51-kDa were studied using mosquito BBMV. Saturation binding, competition binding (homologous and heterologous) and dissociation kinetics studies were carried out using the solubilized, purified, 125I-labeled wild type and mutant binary toxins. All bioassays were carried out in triplicates. Saturation Binding Assay Saturation binding assays were performed by incubating with increasing concentrations (1 to 3 nM) of labeled toxin with fixed quantities (200 g/ml) of BBMV. After 1 h incubation at room temperature, the samples were collected and the amount of toxin bound to the BBMV was calculated by measuring the radioactivity in the pellet (20). The amount of toxin required to achieve saturation binding was similar for both wild type and mutant binary toxins (Fig. 3a). This is in accordance with the earlier reports that all mutants of 42 N-terminal region, which is involved in interaction with the 51-kDa peptide, but not with the receptor, retained toxicity. Whereas the 42
1051
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Line diagram showing the mutations generated at different sites of 51- and 42-kDa peptides of the binary toxin of B. sphaericus by site-directed mutagenesis and their biological activity towards C. quinquefasciatus mosquito larvae. (⫺), Nontoxic up to 10 g/ml protein concentration. (⫹), Toxic.
C-terminal domain (nontoxic mutants 42C4 and 42C5) is not involved in this interaction, lost its biological activity, because of the substitution of crucial amino acids in this region.
recognize the same binding site(s) as that of the wild type toxin. When labeled wild type toxin was competed with excess unlabeled mutant toxins, all the mutants competed as efficiently for the wild type binding site(s) as did unlabeled wild type binary toxin (Fig. 3c).
Competition Membrane Binding Assay In homologous competition binding assays, binding of the labeled toxins was competed with an excess concentration of the corresponding unlabeled toxin to evaluate the binding affinity and affinity concentrations on mosquito larval BBMV. This assay can distinguish between reversible and irreversible binding. Our results showed that the wild type and the mutant binary toxins bind to the BBMV with similar binding affinity (Fig. 3b). Both the wild type and mutant binary toxins bind to the BBMV in a reversible manner. This is in contrast to the binding of Cry toxins of B. thuringiensis, where the binding is mostly irreversible (24). Heterologous competition experiments were performed to evaluate whether the mutant binary toxins
Dissociation Binding Assay Since all the toxins (wild type and mutants) showed similar binding affinities to BBMV, we tested the dissociation kinetics of the BBMV-bound toxins. The labeled toxins were first allowed to bind to the BBMV and were then chased with the corresponding unlabeled toxins. Our results showed that 60 to 80% of the BBMV-bound wild type and mutant toxins were displaced by the addition of excess concentration of unlabeled ligands (i.e., both wild type and mutant binary toxins were reversibly associated), which is shown in the graph (Fig. 3d). The binding assays of wild type and 42-kDa mutant binary toxins against mosquito BBMV show that bind-
1052
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. (a) SDS–PAGE profile of wild type and mutant binary toxins of B. sphaericus 1593M, solubilized in 50 mM sodium carbonate buffer, pH 9. 5. Lanes: 1, molecular weight marker; 3, wild type (pSV15); 4, 42N3; 5, 42N4; 6, 42N5; 7, 42N6; 8, 42C4; 10, 42C5; and 2 & 9, trypsin digested wild type sample. Twenty micrograms of solubilized samples were loaded in each well on a 10% SDS–polyacrylamide gel. (b) SDS–PAGE profile of Trypsin (2% w/w) digested wild type and mutant binary toxins of B. sphaericus 1593M. Lanes: 1, molecular weight marker; 9, wild type toxin (pSV15); and 2, 3, 4, 5, 6, 7, 8, & 10, Mutant toxins (42C6, 42C7, 42N3, 42N4, 42N5, 42N6, 42C4, and 42C5, respectively). Ten micrograms of purified, trypsin digested samples were loaded in each well on a 10% SDS–polyacrylamide gel.
ing is not affected by mutations at the 42-kDa peptide. This result matches with the earlier reports showing the 51-kDa peptide of the binary toxin is involved in binding to the gut receptor (13). However, the biological activity of the binary toxin is decreased or abolished by certain mutations, which were made in the 42-kDa peptide. For example, mutation of 312R residue in the 42 C-terminal completely destroys the biological activity of the binary toxin, even though there is no difference in the binding pattern of this mutant towards mosquito BBMV. The loss of activity of binary toxin
when the residue 312R of 42-kDa was substituted with other positively charged amino acids, viz. lysine and histidine points to the central significance of the arginyl residue possibly in membrane pore formation. This result is in agreement with the previous reports that the C-terminal region of the 42-kDa may be important for pore formation of the toxin. In conclusion, we have identified certain amino acid residues at the N- and C-terminal regions of 51- and 42-kDa peptides that are crucial for the biological activity of the binary toxin. In vitro binding assays with
1053
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. (a) Saturation binding of wild type (pSV15) and mutant (42N3, 42N4, 42N5, 42N6, 42C4, and 42C5) binary toxins of B. sphaericus with C. quinquefasciatus BBMV. (b) Homologous competition binding of wild type (pSV15) and mutant (42N3, 42N4, 42N5, 42N6, 42C4, and 42C5) binary toxins of B. sphaericus with C. quinquefasciatus BBMV. (c) Heterologous competition binding of wild type (pSV15) binary toxin by mutant (42N3, 42N4, 42N5, 42N6, 42C4, and 42C5) toxins of B. sphaericus with C. quinquefasciatus BBMV.* 125I-labeled toxins. (d) Dissociation kinetics of wild type (pSV15) and mutant (42N3, 42N4, 42N5, 42N6, 42C4, and 42C5) binary toxins of B. sphaericus with C. quinquefasciatus BBMV.
1054
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
other mutants of 51 N- and C-terminal region will further enable us in understanding the mode of action of this toxin. Further, the role of residues 327R and 303E towards biological activity and binding to BBMV is under investigation. ACKNOWLEDGMENTS We thank April Curtiss of the Department of Biochemistry, Ohio State University, Columbus, Ohio, and Dr. Geetha of Centre for Biotechnology, Anna University, Chennai, India, for their help and discussion of this work. We acknowledge Indo-Swiss Collaboration in Biotechnology, Switzerland, Council of Scientific and Industrial Research, New Delhi, India, and Department of Biotechnology, Government of India, for financial support of the project.
REFERENCES 1. Baumann, P., Clark, M. A., Baumann, L., and Broadwell, A. H. (1991) Microbiol. Rev. 55, 425– 436. 2. Porter, A. G., Davidson, E. W., and Liu, J. W. (1993) Microbiol. Rev. 57, 838 – 861. 3. Shanmugavelu, M., Elangovan, G., Kathirvel, M., and Jayaraman, K. (1998b) Biotech. Lett. 20(4), 341–344. 4. Berry, C., Jackson-Yap, J., Oei, C., and Hindley, J. (1989) Nucleic Acids Res. 17, 7516. 5. Davidson, E. W. (1988) J. Med. Entomol. 25, 151–157. 6. Charles, J. -F., Nielson-LeRoux, C., and Delecluse, A. (1996) Ann. Rev. Entomol. 42, 451– 472. 7. Broadwell, A. H., Clark, M. A., Baumann, L., and Baumann, P. (1990) J. Bacteriol. 172, 4032– 4036. 8. Clark, M. A., and Baumann, P. (1990). J. Bacteriol. 172, 6759 – 6763.
9. Sebo, P., Bennardo, D., De la Torre, F., and Szulmajster, J. (1990) Eur. J. Biochem. 194, 161–165. 10. Oei, C., Hindley, J., and Berry, C. (1992) J. Gen. Microbiol. 138, 1515–1526. 11. Nielsen-LeRoux, C., and Charles, J. F. (1992) Eur. J. Biochem. 210, 585–590. 12. Silva-Filha, M. H., Nielsen-LeRoux, C., and Charles, J. -F. (1997) Eur. J. Biochem. 210, 585–590. 13. Silva-Filha, M. H., Nielsen-LeRoux, C., and Charles, J. -F. (1999) Insect Biochem. Mol. Biol. 29, 711–721. 14. Shanmugavelu, M., Rajamohan, F., Kathirvel, M., Elangovan, G., Dean, D. H., and Jayaraman, K. (1998a) Appl. Environ. Microbiol. 64, 756 –759. 15. Shanmugavelu, M., Meenakshisundaram, B., and Jayaraman, K. (1997) Biotechnol. Lett. 19, 341–344. 16. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488 – 492. 17. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463–5467. 18. Almond, B. D., and Dean, D. H. (1993) Appl. Environ. Microbiol. 59, 2442–2448. 19. Lowry, O. H., Rosebrough, N. H., Farr, N. J., and Randal, R. J. (1951) J. Biol. Chem. 193, 265–268. 20. Rajamohan, F., Alcantara, E., Lee, M. K., Chen, X. J., Curtiss, A., and Dean, D. H. (1995) J. Bacteriol. 177, 2276 –2282. 21. Wolfersberger, M. G., Luthy, P., Maurer, A., Parenti, P., Sacchi, F. V., Gioordana, B., and Hanozet, G. M. (1987) Comp. Biochem. Biophysiol. 86, 301–308. 22. de Barjac, H. (1990) Rutgers University Press, Brunswick, N J. 23. Davidson, E. W., Oei, C., Meyer, M., Beiber, A. L., Hindley, J., and Berry, C. (1990) Can. J. Microbiol. 36, 870 – 878. 24. Liang, Y., Patel, S. S., and Dean, D. H. (1995) J. Biol. Chem. 270, 24719 –24724.
1055