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Nucleotide sequence of the gene coding for a 130-kDa mosquitocidal protein of Bacillus thuringiensis israelensis

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Nucleotide sequence of the gene coding for a 130-kDa mosquitocidal protein of Bacillus thuringiensis israelensis
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  Gene, 66 (1988) 107-120 Elsevier 107 GEN 02405 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Nucleotide sequence of the gene coding for a 130-kDa mosquitocidal protein of Bacillus thzwingiensis israelensis (Recombinant DNA; plasmid; endotoxin; crystal protein) T. Yamamotoa*b, .A. Watkinson”-b, L. Kimb>*, M.V. Sage’, R. Strattonc, N. Akande’, Y. Li’, D.-P. Ma’ and B.A. RoeC zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA “Agricultural Products Department, DuPont Company, 402 Experimental Station, Wilmington, DE 19898 U.S.A.): bShell Development Company, Modesto, CA 95352 U.S.A.) Tel. 209) 545-8100, and’ Chemistry Department, University of Oklahoma, Norman, OK 73019 U.S.A.) Tel. 405) 325-4912 Received 30 December 1987 Accepted 7 January 1988 Received by publisher 29 February 1988 SUMMARY The nucleotide sequence of pVB 13 1 containing the gene coding for a 130-kDa Bacillus thuringiensis israelensis B. t.isr) mosquitocidal protein was determined. The pVB131 plasmid was constructed by Sekar and Carlton [Gene 33 (1985) 151-1581. Our sequencing revealed only one open reading frame large enough to code for a protein of 130 kDa. The translation start site was determined by sequencing the protein isolated from B.t.isr. The amino acid sequence of the protein was deduced from the nucleotide sequence, and its M, was determined as 128505. Immunological and biochemical analyses of B.t.isr mosquitocidal proteins indicated that the 130-kDa protein coded by pVB131 was indeed expressed in B.t.isr. Comparing the peptide sequence of the 130-kDa B.t.isr toxin with the sequences of other B.t. toxins having activities specific to lepidopteran species showed that several domains were highly homologous. This suggests that they are evolutionarily related to each other, and in the evolutionary process the sequences in the homologous domains that are important to the insecticidal activity have been conserved. INTRODUCIION Bacillus thuringiensis is known as one of a few commercially available microbial insecticides. When B. t. is cultured in a medium suitable for supporting its growth, it produces a spore and one or more crystals in each cell after the stationary phase is reached. Each crystal is made of one or more pro- teins which are in most cases insecticidal. B.t.isr which was discovered in Israel (Goldberg Correspondence o: Dr. T. Yamamoto, Agricultural Products De- partment, DuPont Company, 402 Experimental Station, Wilmington, DE 19898 (U.S.A.) Tel. (302) 695-4372. * Present address: Mycogen Corporation, 5451 Oberlin Drive, San Diego, CA 92121 (U.S.A.) Tel. (619) 453-8030. Abbreviations: aa, amino acid(s); bp, base pair(s); B.t., zyxwvutsrqpon acillus thuringiemi~; sr, isruelensh; kb, 1000 bp; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; SDS, sodium dodecyl sulfate. 037X-l 119/88/ 03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)  108 and Margalit, 1977) produces at least three, perhaps four proteins in three different crystal forms (i.e., three differently shaped crystals can be seen in one bacterial cell) (Yamamoto et al., 1983; Ibara and Federici, 1986; Federici et al., 1987). M,s of these proteins were estimated as 130000-135000 and 65 000 by SDS-PAGE (Wu and Chang, 1985; Ibara and Federici, 1986; Federici et al., 1987) and 27 340 by cloning and sequencing one of the genes (Waalwijk et al., 1985; Ward et al., 1986). There are a number of contradictory reports on the mosquito- tidal activities of the B. t.isr crystal proteins, but we found that, as reported by Wu and Chang (1985), all individual proteins were mosquitocidal and the toxi- city was remarkably enhanced by mixing two or more of these proteins. It has been shown that SDS-PAGE resolves the B.t.isr protein or proteins of about 130 kDa into two bands (Thomas and Ellar, 1983; Federici et al., 1987). The gene coding for one of the 130-kDa B. t.isr toxins has been cloned by Sekar and Carlton (1985). To clone the gene, they constructed a plasmid designated pVB 131 which contained three XbaI fragments of B.t.isr plasmid DNA. When the pVB 13 1 plasmid was introduced to Bacillus megaterium, the transformed B. megaterium termed VB131 produced a mosquitocidal crystal (Sekar and Carlton, 1985). Our present paper describes the sequence of the cloned gene in the pVB 13 1 plasmid and comparisons of the sequence of this B.t. gene with those of other B.t. toxin genes. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA MATERIALS AND METHODS a) Protein isolation and characterization The B.t.isr strain HD567-61-9, which contains only the 114-kb plasmid or ‘75-MDa’ plasmid as described by Gonzalez and Carlton (1984), was obtained from Dr. Carlton. Production and isolation of the proteinase-free crystals has been described previously (Yamamoto, 1983). The 130-kDa pro- teins were extracted from the crystals of B. t.isr and B. megaterium VB13 1 with 2% 2-mercaptoethanol at pH 10 adjusted with 2 N NaOH. The extract was centrifuged at 50000 x g for 30 min and the super- natant was chromatographed in a Sephacryl S-300 column. The 130-kDa protein eluted from the col- umn at an elution volume of about 240 ml (see Fig. 1) was directly charged onto a DEAE-Sepharose col- umn. The isolated 130-kDa protein or proteins were eluted from the column with a 0.1-0.25 M NaCl gradient at 0.2 M, dialyzed in water, and lyophilized. Purity of the protein was examined by immuno- electrophoresis and SDS-PAGE. Antiserum was made in rabbits using solubilized crystals isolated from B. t.isr. The N-terminal amino acid sequence of the protein was determined with a Beckman System 890 protein/peptide sequencer. b) Nucleotide sequencing The recombinant plasmid, pVB131, and its B. megaterium host, VB131, (Sekar and Carlton, 1985) were provided by Drs. V. Sekar and B.C. Carlton. The B. t.isr DNA was inserted into the XbaI site of pBC16 from Bacillus cereus (Bernhard et al., 1978). This chimeric plasmid was cleaved into four fragments with XbaI and subcloned in the XbaI site of M13mp8 (Messing et al., 1981) to generate four complementary sets of single-stranded chimeric phage DNAs encompassing the entire recombinant plasmid (Messing and Vieira, 1982). Double- stranded chimeric phage DNA was isolated as de- scribed (Messing, 1983). The clonedXba1 fragments were excised by digestion with XbaI, purified by pre- parative 0.8% agarose gel electrophoresis, eluted by a modified freeze-thaw method, and concentrated by electrophoresis. After further digestion with selected restriction endonucleases, fragments of pVB 13 1 were ligated into either M13mp8, M13mp9, M13mp10, M13mpl1, M13mp18, or M13mp19 (Messing and Vieira, 1982; Messing, 1983; Yanisch- Perron et al., 1985). In some instances, large frag- ments, which lacked appropriate restriction sites, were gel-purified and treated with exonuclease BAL 3 1 for various time intervals prior to ligation into M13mp9 to obtain overlapping segments. The single-stranded recombinant phage DNAs contain- ing fragments of pVB 131 were isolated by phenol extraction of the polyethylene glycol-concentrated phage (Sanger et al., 1980). All nucleotide sequence data were obtained by the dideoxynucleotide chain-termination method (San- ger et al., 1977; 1980). The Ml3 subclones were selected first at random and later by hybridization to  109 zyxwvutsr 16 24 32 Elution volume ml) zyxwvutsrqponmlkjihgfedcbaZYXWVUTS 130 67 --A y; * . : ..* ..--- .m.m.. +L TYti131 protein 00MG~,00~,0000 000‘000’000’0000 00~>00000000C>00 006000000000000 I I I I . I I a I I 240 320 Fig. 1. Immunological compatibility between two 130-kDa proteins produced in B.t.isr and B. meg terium VB131. Panel A shows a partial separation of the B.t.isr toxins by Sephacryl S-300 column chromatography. B.t.isr crystals were dissolved in an alkaline solution and proteins were separated in a 2.5 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB 95-cm column of Sephacryl S-300. In panels B and C, the column eluate was analyzed by fused-rocket immunoelectrophoresis. The electrophoresis was conducted on 10 x IO-cm glass plates by following the protocol described by Svendsen (1973). Agarose (1.2%) containing the antiserum (2.5 pi/ml) which had been made in rabbits against total B.t.isr crystal proteins was poured on the glass plates in l-mm thick layers. The column eluate from 180 to 375 ml was fractioned in g-ml aliquots, and 491 samples of the fractions were placed in individual wells punched in two rows on the bottom edge of the agarose gels. In panel C, part of the gel, a 7-mm wide and IO-cm long strip marked ‘VB 131 protein’, contained the VB 131 protein at 0.1 mg/ml. The column eluate in the wells was allowed to diffuse into the gel for 1 h and electrophoresed at 100 V for 16 h. After the electrophoresis, the gel was washed in 0.5% NaCl to remove the serum proteins; the antigen-antibody complex (precipitin lines) appearing as peaks was stained with 0.25 y0 Coomassie blue, and density of the stain was digitized with an Apple Macintosh equipped with a scanner at a resolution of 30 points per cm.  110 overlapping fragments cloned in M 13 in the opposite orientation. All inserts were sequenced using a syn- thetic oligodeoxynucleotide primer. The complete sequence of contiguous regions was assembled from individual overlapping subclones containing frag- ments in either orientation, using the programs de- scribed by Staden (1982). All regions of pVB131 were sequenced at least twice in one orientation and over 90% was sequenced in both orientations to remove discrepancies. The sequencing strategy and partial restriction map are shown in Fig. 2. c) Computer analysis Nucleotide and amino acid sequences were ana- lyzed by computers using the University of Wisconsin Genetic Computer Group program (Devereux et al., 1984) installed on a VAX 11/780 and the DNA Inspector II (Textco, West Lebanon, NH) on an Apple Macintosh. RESULTS AND DISCUSSION a) The 130-kDa protein expressed in Bacillus thuringiensis israelensis When the 130-kDa B.t.isr toxin gene (130-kDa B.t.isr gene) was cloned by Sekar and Carlton (1985), the 114-kb or ‘75-MDa’ plasmid of B.t.isr was digested by XbaI, and three X&I fragments were ligated in a series with a cloning vector, pBC16. Since no XbaI fragment is large enough to code for a protein of 130 kDa and there may be two highly homologous genes coding for 130-kDa toxins, it is possible that the coding region of pVB 13 1 is a hybrid of two pieces of different genes. We, however, con- sider that this is unlikely. Bourgouin et al. (1986) cloned in Escherichia coli a lo-kb EcoRI fragment from a strain of B.t.isr containing only the 109-kb or ‘72-MDa’ plasmid. The clone containing the lo-kb EcoRI fragment in a recombinant plasmid termed pRX8 produced a 130-kDa mosquitocidal protein. According to the restriction map of pRX8 published by Bourgouin et al. (1986), the 5’ region encompass- ing sites from EcoRI to XbaI (approx. 3.4 kb) of the lo-kb EcoRI fragment appeared to be the same as the 3 ’ region of the B. t. isr DNA of pVB 13 1. It has been reported that B. t. kurstaki HD-1 contains several insecticidal toxin genes one of which is not expressed (Kronstad and Whiteley, 1986; Yamamoto et al., 1988). We, however, concluded from our present study that the coding region of pVB 13 1, whether it is a hybrid of two genes or not, contains at least a part of a B.t.isr gene coding for a 130-kDa protein. In B.t.isr, this gene is expressed at a level high enough to form the crystal. Expression of this gene in B. t. isr was confirmed by two methods : (i) immunochemical compatibility between a native 130-kDa protein from B.t.isr and the 130-kDa pro- tein coded by pVB131 and produced in B. megate- rium VB13 1; (ii) isolation and sequencing of a 130-kDa protein produced by B. t.isr. Fig. 1 is a computer image of two immunoelectro- phoretograms showing that the gene product of pVB 13 1 (VB 13 1 protein) neutralized the antibodies directed to a B.t.isr 130-kDa protein. Sekar (1986) reported that the B. megaterium VB131 containing pVB13 1 produced 130-, 96-, 85-, and 40-kDa pro- teins positively reacting with antiserum directed to the total B.t.isr crystal proteins. We prepared the crystals from VB 13 1 after the spore-crystal complex was extensively washed in 0.5 M NaCl to remove the cellular proteinases and found that the amounts of the proteins other than 130 kDa were greatly reduced to a level barely detectable with Coomassie dye. In Fig. lA, the three B.t.isr proteins of 27, 67, and 130 kDa were partly separated by Sephacryl S-300 column chromatography. The antiserum recognized these proteins as different proteins which appeared as three independent peaks (Fig. 1B) termed precipi- tin lines (Svendsen, 1973). In the other electro- phoresis (Fig. lC), the VB 13 1 protein was placed in a 7-mm strip across the lower part of the gel to see which B.t.isr protein would cross-react with it. As shown in Fig. lC, the precipitin line of the VB 13 1 protein fused with the line of a 130-kDa B.t.isr pro- tein but not with those of the 27- and 67-kDa B.t.isr proteins. It was observed that a weak but distinctive precipitin line (indicated by a white arrow) appeared after the antibodies directed to a 130-kDa B.t.isr protein were neutralized by the VB 13 1 protein. This observation suggests that B. t.isr produces two pro- teins having A4,s in the vicinity of 130 kDa.  111 6 6.45 I---- - -+ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA  w-t-( pBC16 t- ------_ -+t+ _3 - -+ -- -M-u c--d -----_)---_) * ; z ++ Q 3 Fig. 2. Sequencing strategy and selected restriction sites presented on a partial map ofpVB131. Arrows below represent the segments sequenced and the direction of the sequencing. The protein-coding region (ORF) is shown as a stippled box encompassing from 2.92 kb to 6.32 kb. The open boxes represent the flanking regions of the B.t.isr gene coding for the 130-kDa toxin. The blackened boxes represent the pBC16 vector. b) Nucleotide sequence of pVB131 We sequenced the entire 6446-bp B.t.isr DNA insert in pVB 13 1 (Fig. 2). Fig. 3 shows a part of the nucleotide sequence of pVB 131 which includes a large ORF. This ORF resides in the 3’ end of the B.t.isr DNA inserted into pBC16 (Fig. 2). In the 5’ end of the ORF, there were several possible trans- lation start sites for a protein of about 130 kDa. To determine the translation start site, the 130-kDa pro- tein was isolated from B.t.isr crystals and its N-ter- minal amino acids were sequenced. This experiment was also necessary to confirm the expression of this gene in B.t.isr. The protein isolation was performed by two consecutive column chromatography separa- tions, first with Sephacryl S-300 followed by DEAE- Sepharose. SDS-PAGE conducted with a 7.5% gel containing 0.1% SDS showed that the purified sample contained no proteins other than 130 kDa. Sequencing the isolated protein revealed the N-ter- minal amino acid sequence (Fig. 3, underlined) that was found on a translation starting at nt 2916. Our amino acid sequencing, however, indicated that there was another amino acid residue at each sequencing step. The second sequence had considerable ambi- guity due to the small amount of the protein but was partly determined as (M)-E-Q-F-X--- (X, unidenti- fied). The second amino acid residues, although they were minor components, suggested that the B. t.isr crystals contain an additional 130-kDa protein gene which was expressed in a lesser amount. The minor component found in the preparation of the 130-kDa protein could be one of two bands as shown in SDS-PAGES in previous reports (Thomas and Ellar, 1983; Federici et al., 1987). Ward and Ellar (1987) published a short note showing the nucleotide sequence of a B.t.isr gene that codes for a 135-kDa protein. Our SDS-PAGE, however, failed to resolve the crystal proteins into two bands around 130 kDa. Furthermore, the N-terminal amino acid sequence of the 135kDa protein deduced from the nucleotide sequence (Ward and Ellar, 1987) was different from the N-terminal amino acid sequences of the 130-kDa proteins isolated from our B.t.isr strain, HD567- 61-9. We, therefore, concluded that, in HD567-61-9, the gene coding for the 135-kDa protein was not expressed and the expression level of the second 130-kDa gene was very low. It was evident that the sequenced DNA, at least the second XbaI fragment containing the translation start site, came from the B. t.isr gene coding for a 130-kDa toxin. The coding region starts with ATG at nt 2916 and ends with TGA at nt 6321. It codes for a protein of n/l, 128505 consisting of 1135 aa residues. We found an inverted repeat sequence in the 3’-flanking region (Fig. 3, facing arrows). When the DNA was transcribed into mRNA, this inverted repeat could form a stable ‘hairpin structure’ having a 16-nt stem and a 6-nt loop. The inverted repeat could serve as the transcription terminator for the 130-kDa B.t.isr gene, but further study is required to confirm this. Further analyses on the nucleotide sequence were done in conjunction with other B. t. toxin gene sequences as follows.
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