APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/00/$04.0010

Oct. 2000, p. 4449–4455 Vol. 66, No. 10

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Molecular Genetic Manipulation of Truncated Cry1C Protein Synthesisin Bacillus thuringiensis To Improve Stability and Yield

HYUN-WOO PARK,1 DENNIS K. BIDESHI,1 AND BRIAN A. FEDERICI1,2,3*

Department of Entomology1 and Interdepartmental Graduate Programs in Genetics2 and Microbiology,3

University of California, Riverside, Riverside, California 92521

Received 18 April 2000/Accepted 13 July 2000

Cry1 protoxins of Bacillus thuringiensis are insecticidal 135-kDa proteins synthesized and assembled intoparasporal crystals during sporulation. After ingestion, these crystals dissolve in the midgut and active toxinswith molecular masses of about 65-kDa are released from the N-terminal half of the molecule by midgutproteases. Direct synthesis of the toxin-containing N-terminal half of Cry1 molecules using recombinant DNAtechniques results in a low level of unstable truncated proteins that do not crystallize. In the present study,inclusions of truncated Cry1C (Cry1C-t) were obtained by combining genetic elements from other endotoxingenes and operons that enhance Cry protein synthesis and crystallization. Increased levels of Cry1C-t synthesiswere achieved by using cyt1A promoters to drive expression of the 5* half of cry1C that included in the constructthe 5* cry3A STAB-SD mRNA stabilizing sequence and the 3* stem-loop transcription terminator. RNA dot blotanalysis showed that the STAB-SD and 3* transcriptional termination sequences were important for stabili-zation of truncated cry1C (cry1C-t) mRNA. A low level of cry1C-t mRNA was present when only the cyt1Apromoters were used to express cry1C-t, but no accumulation of Cry1C-t was detected in Western blots. Theorientation of the transcription terminator was important to enhancing Cry1C-t synthesis. Inclusion of the 20-and 29-kDa helper protein genes in cry1C-t constructs further enhanced synthesis. The Cry1C-t protein wastoxic to Spodoptera exigua larvae, though the toxicity (50% lethal concentration [LC50] 5 13.2 mg/ml) was lowerthan that of full-length Cry1C (LC50 5 1.8 mg/ml). However, transformation of the HD1 isolate of B. thurin-giensis subsp. kurstaki with the cry1C-t construct enhanced its toxicity to S. exigua as much as fourfold.

Insecticidal Cry proteins produced by Bacillus thuringiensisare the principal active ingredients of most bacterial insecti-cides. Based on mass, there are two major types of Cry pro-teins, those with molecular masses of approximately 135 kDa,such as the common Cry1 protoxins, and those with molecu-lar masses of approximately 70 kDa, exemplified by Cry2A,Cry3A, and Cry11A (16). The amino acid sequence of thelatter type corresponds to the amino acid sequence of theN-terminal half of the former type. Cry proteins typically aresynthesized as protoxins during sporulation and are assembledinto crystals that stabilize the toxin (3, 8). When ingested byinsects, the crystals dissolve in the midgut and the protoxin iscleaved by midgut proteases, releasing an active polypeptidewith a molecular mass of 65 to 68 kDa (1, 16, 17).

Because the C-terminal half of 135-kDa Cry1 protoxins isnot toxic, if it could be eliminated and the cellular resourcescould be redirected to synthesize an equivalent additionalamount of the N-terminal half, the specific toxicity—i.e., thetoxicity per unit of mass of bacterial insecticides—might beimproved. This would in essence convert Cry1 proteins bytruncation into toxins like Cry2A or Cry3A. When truncatedcry1 genes are expressed in B. thuringiensis, however, the toxinyields are low, and the truncated proteins do not form inclu-sions (1, 15, 22). Possible reasons for this include a low level oftruncated gene expression, instability of the truncated mRNAand protein, and inefficient crystallization of the truncatedprotein.

Several genetic elements that enhance synthesis and crystal-lization of “naturally truncated” Cry toxins, such as Cry2A,

Cry3A, and Cry11A, have been identified recently. These ele-ments include the 59 STAB-SD mRNA stabilizing sequence ofcry3A (2), 39 stem-loop structures that also stabilize cry tran-scripts (24), and two helper proteins that enhance translationand/or crystallization. These two helper proteins are the 20-kDa chaperone-like protein encoded by orf3 of the cry11Aoperon (6, 25, 26) and the 29-kDa protein encoded by orf2 ofthe cry2A operon (23), which apparently serves both as a mo-lecular chaperone (5) and a scaffolding protein that facilitatesCry2A crystal formation (7).

Several studies have shown that these elements, alone or incombination with each other or other genetic elements, can bemanipulated to enhance Cry synthesis. For example, Park et al.(13) demonstrated that Cry3A synthesis could be increasedmore than 10-fold in comparison to the synthesis by the wild-type strain by driving expression of cry3A, including the STAB-SD sequence, with strong cyt1A promoters. In another study,synthesis of Cry2A and Cry11A using the cyt1A promoters–STAB-SD expression system resulted in more moderate butstill significant increases of 4.4- and 1.3-fold, respectively (14).With respect to the helper proteins, the 20-kDa protein en-hances net synthesis of Cry2A (7).

In the present study, we made various combinations of theseenhancer elements and evaluated them to determine theircapacities to enhance the synthesis and crystallization of trun-cated Cry1C (Cry1C-t) molecules. Here we show that inclu-sions of Cry1C-t can be produced by combining mRNAstabilizing sequences and helper protein genes in constructscontaining truncated cry1C (cry1C-t). We also show that addingthis construct to a wild-type strain of B. thuringiensis improvesits insecticidal activity against the beet armyworm, Spodopteraexigua, an important insect pest.

* Corresponding author. Mailing address: Department of Entomol-ogy, University of California, Riverside, CA 92521. Phone: (909) 787-5006. Fax: (909) 787-3086. E-mail: brian.federici@ucr.edu.

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MATERIALS AND METHODS

Bacterial strains and transformation. Plasmid constructs were amplified inEscherichia coli DH5a. The cry1C constructs were expressed in an acrystallifer-ous strain of B. thuringiensis subsp. israelensis (4Q7), B. thuringiensis subsp.kurstaki HD-1, or B. thuringiensis subsp. aizawai 1857. The B. thuringiensis strainswere transformed by electroporation as previously described (13).

PCR. The PCR was performed by using the Expand Long Template PCRsystem (Boehringer, Mannheim, Germany) or with Vent (Exo1) DNA polymer-ase (New England Biolabs).

Construction of cry1C-t. The plasmid constructs used are shown in Fig. 1 andTable 1, and the primers used for gene amplification are listed in Table 2.Plasmid p2-44, which contained the intact cry1C gene (GenBank accession num-ber X96682), was provided by Abbott Laboratories (North Chicago, Ill.). Thecry1C gene was obtained as a 4.8-kb EcoRI-HindIII fragment from p2-44, filledwith the Klenow fragment, and cloned into the SmaI site of pHT3101 (11) togenerate pPF1C. The cry1C open reading frame (ORF) in pPF1C was amplifiedwith primers 1Ca-1 and 1Ca-3 (Table 2) by using a DNA thermal cycler (Perkin

FIG. 1. Summary of vector construction for expression of full-length and truncated cry1C genes. (A) The vectors pPFT3As and pPF1C were used as templates forfurther construction. The full-length cry1C gene, including its promoter region, was obtained as a 4.8-kb fragment of plasmid 2-44 (see Materials and Methods) partiallydigested with EcoRI and HindIII. This fragment was treated with the Klenow fragment and inserted into pHT3101 to generate pPF1C. (B) The full-length and truncatedcry1C ORFs beginning at the ATG codon were inserted into the SalI-SphI sites and SalI sites of pPF-CH (see Materials and Methods) to generate pPFT1Cs andpPFT1Cs-t, respectively. For cry1C-t without the STAB-SD sequence, the same fragment used for construction of pPFT1Cs-t was inserted into SalI sites of pHTCytA.To add the 39 TTS to truncated cry1C-t, a 479-bp cry3A termination sequence was inserted into the SphI site of pPFT1Cs-t [pPFT1Cs-3t(1) and pPFT1Cs-3t(2)]. (C)The vectors pPFT2Asf and pPFT11Ast were used as templates for amplification of orf2 and the 20-kDa protein gene. (D) For the 20-kDa protein, a 1.5-kb fragmentfrom pPFT11Ast was inserted into the SphI site of pPFT1Cs-t (pPFT1Cs-20k). For the ORF2 protein gene, a 850-bp fragment from pPFT2Asf was inserted into theXbaI-SalI site of pPF-CH (pPF-ORF2). Then 2.38- and 3.41-kb fragments, obtained from SalI-SphI partial digestion of pPFT1Cs-3t(1) and pPFT1Cs-20k, respectively,were inserted into the SalI-SphI site of pPF-ORF2 to generate pPFT1Csf-3t(1) and pPFT1Csf-20k.

TABLE 1. Summary of genetic elements presentin each cry1C construct

Construct Genetic elements presenta

pPF1C.......................cry1C p 1 full-length cry1CpPFT1Cs...................cyt1A p 1 STAB-SD 1 full-length cry1CpPFT1C-t .................cyt1A p 1 cry1C-tpPFT1Cs-t ................cyt1A p 1 STAB-SD 1 cry1C-tpPFT1Cs-3t(1) .......cyt1A p 1 STAB-SD 1 cry1C-t 1 stem-loop (59-39)pPFT1Cs-3t(2) .......cyt1A p 1 STAB-SD 1 cry1C-t 1 stem-loop (39-59)pPFT1Csf-3t(1) ......cyt1A p 1 STAB-SD 1 orf2 1 cry1C-t 1 stem-loop

(59-39)pPFT1Cs-20k ...........cyt1A p 1 STAB-SD 1 cry1C-t 1 20-kDa protein genepPFT1Csf-20k..........cyt1A p 1 STAB-SD 1 orf2 1 cry1C-t 1 20-kDa

protein gene

a p, promoters (i.e., multiple promoters for all constructs listed).

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Elmer GeneAmp PCR system 2400) and inserted into expression vector pPF-CHthat contained the cyt1A promoters and STAB-SD sequence to generatepPFT1Cs. The cry1C-t fragment was amplified with primers 1Ca-1 and 1Ca-2 andinserted into pHTCytA (13), which contained cyt1A promoters, to generatepPFT1C-t or into pHTCytA with STAB-SD (pPF-CH) to generate pPFT1Cs-t.The cry1C-t gene encodes 630 amino acids, and the C-terminal amino acid isK630. The 479-bp fragment containing the transcription termination sequence(TTS) in pPFT3As (13) was obtained by PCR using primers 3Aa-1 and 3Aa-2.The 479-bp fragment was digested with SphI and cloned into the same site inpPFT1Cs-t to generate pPFT1Cs-t3(1), with the TTS in the same orientation asthe cry1C ORF, and pPFT1Cs-3t(2), with the TTS in the orientation oppositethat of the cry1C ORF. The 1.5-kb fragment in pPFT11Ast containing the20-kDa gene was amplified with primers 11Aa-1 and 11Aa-2 and inserted intothe SphI site of pPFT1Cs-t to generate pPFT1Cs-20k. The 850-bp fragment inpPFT2Asf containing orf2 of the cry2A operon was amplified with primers 2Aa-1and 2Aa-2 and inserted into the XbaI-SalI site of pPF-CH to generate pPF-ORF2. The 2.38- and 3.41-kb SalI-SphI fragments were obtained from pPFT1Cs-3t(1) and pPFT1Cs-20k, respectively, and were inserted into the SalI-SphI siteof pPF-ORF2 to generate pPFT1Csf-3t(1) and pPFT1Csf-20k.

cry1C-specific antisense probe. A 0.8-kb cry1C-specific antisense DNA probewas made by unidirectional PCR using digoxigenin-labeled nucleotides (Boehr-inger) and the 1Ca-2 primer. The truncated cry1C-t gene PCR product was usedas the substrate for Vent (Exo1) DNA polymerase (New England Biolabs).

RNA isolation and dot blot analysis. RNA was isolated from sporulating cellsgrown in 10 ml of nutrient broth plus salts (NBG) at 30°C for 12 h (14). Thebacterial cells were centrifuged at 6,000 3 g for 5 min at 4°C, and the pellets weresuspended in 1 ml of TRIzol reagent (GIBCO BRL, Grand Island, N.Y.).Sodium dodecyl sulfate (SDS) was added to a final concentration of 1% (vol/vol).The suspension was sonicated 10 times on ice at 50% duty cycle for 15 s(Ultrasonic Homogenizer 4710 series; Cole-Parmer Instrument Co., Chicago,Ill.). After samples were incubated at room temperature for 5 min, 200 ml ofchloroform was added. The samples were mixed thoroughly and centrifuged at12,000 3 g for 15 min. The aqueous phase was transferred to a fresh tube, and500 ml of isopropanol was added. After incubation at room temperature for 10min, RNA was collected by centrifugation at 12,000 3 g for 10 min. The RNApellets were washed with 1 ml of 75% ethanol, spun at 7,500 3 g for 5 min, dried,and dissolved in 50 ml of diethyl pyrocarbonate-treated double-distilled water.RNA concentrations were determined by measuring the absorption at 260 nmwith a PM6 spectrophotometer (Zeiss, Oberkochen, Germany). RNA samples(1 and 2.5 mg) were spotted on a nylon membrane (Micron Separations, Inc.,Westborough, Mass.). The membrane was dried in a vacuum oven at 80°C for2 h. Prehybridization and hybridization at 42°C with the antisense-cry1C DNAprobe and detection with the CDP-Star reagent (Boehringer) were performedaccording to the manufacturer’s protocol. After exposure, the detection film wasscanned with the GAS 4000 gel documentation system (Evergreen). The level ofhybridization was quantified by using ImageQuant 4.1 densitometry software(Molecular Dynamics, Sunnyvale, Calif.). Hybridization values were determinedby comparison to the signal obtained with reference plasmid pPFT1C-t, whichwas assigned a value of 1. RNA dot blots were replicated three times by usingthree different RNA preparations from three different cultures. Data from theblots were analyzed with the Super ANOVA program (Abacus Concepts, Berke-ley, Calif.) (13, 14).

SDS-PAGE. Bacterial strains were grown in 50 ml of NBG (14) at 30°C for 5days, by which time the cells had sporulated and lysed. Spores, crystals, and celldebris were pelleted in 1.2-ml aliquots by centrifugation at 15,000 3 g for 1 min.The pellets were suspended in 50 ml of 53 sample buffer (10) and boiled for 5min. After centrifugation at 15,000 3 g for 5 min to remove solids, 10-ml sampleswere loaded onto a 12% polyacrylamide gel and the proteins were separated byelectrophoresis. Samples taken from three sets of cultures grown on differentdays were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), and thegels were scanned and quantified with the Super ANOVA program (AbacusConcepts) (13, 14).

Purification of Cry1C inclusions. Sporulated cells were pelleted by centrifu-gation at 6,500 3 g for 15 min, suspended in 15 ml of distilled water, andsonicated twice at 50% duty cycle for 15 s by using the Ultrasonic Homogenizer4710. Five-milliliter samples were loaded onto a discontinuous NaBr gradient(9), which was then centrifuged at 20,000 3 g for 45 min at 10°C in a BeckmanL7-55 ultracentrifuge. Bands containing inclusions were collected and dialyzed inwater overnight at 4°C. Purified inclusions were pelleted and lyophilized.

Western blot analysis. Protein concentrations were determined by the methodof Bradford (4). Proteins in 5- or 10-mg samples were separated by electrophore-sis in an SDS–10% polyacrylamide gel and electroblotted onto a polyvinylidenedifluoride membrane (Micron Separations, Inc.) by using a model PS50 elec-troblotter (Hoefer Scientific Instruments). Western blot analysis was performedby using primary rabbit anti-Cry1C antibody kindly provided by W. J. Moar(Department of Entomology, Auburn University, Auburn, Ala.) and alkalinephosphatase-conjugated goat anti-rabbit immunoglobulin G (Southern Biotech-nology Associates, Inc., Birmingham, Ala.) as the secondary antibody (20). Bind-ing of the secondary antibody was detected with the nitroblue tetrazolium and5-bromo-1-chloro-3-indolyl phosphate (BCIP) reagents (Promega, Madison,Wis.).

Microscopy. Sporulating cultures were monitored and photographed with aDMRE phase-contrast microscope (Leica) at a magnification of 31,000.

Bioassays. Bioassays with neonate S. exigua larvae were carried out as previ-ously described (12). The assays were performed in 24-well plates (Corning) byusing lyophilized powder preparations containing spores and crystal inclusionsmixed in artificial diet, as described by Moar et al. (12). Two larvae were placedin each well and then held at 28°C under a daily regime consisting of 16 h of lightand 8 h of darkness. A total of 48 larvae were used for each protein concentrationassayed. Larval mortality was determined after 7 days of exposure to the spore-crystal toxin mixture; larvae were considered to be alive if they were able torespond to tactile stimulation.

RESULTS

Expression of cry1C-t. When cry1C-t was expressed usingpPFT1C-t (Fig. 1), which contained cyt1A promoters to driveexpression but lacked the STAB-SD and cry3A 39 stem-loopsequences, the transcript level as determined by RNA dot blotswas low (Fig. 2, lane 1). Inclusion of STAB-SD in the construct(pPFT1Cs-t) yielded a twofold increase in the level of cry1C-ttranscript detected (Fig. 2, lane 2). When both STAB-SD andthe 39 stem-loop were included in the construct [pPFT1Cs-3t(1)], the transcript level increased to 2.6 times that of theconstruct which lacked these elements, pPFT1C-t (Fig. 1; Fig.2, lane 3; Table 1). Placement of the cry3A stem-loop in theorientation opposite that of cry1C-t [pPFT1Cs-3t(2)] (Fig. 1;Table 1) resulted in a marked reduction in the level of tran-script detected (Fig. 2, lane 4).

No significant differences were found between the cry1C-ttranscript levels with constructs that contained the 29-kDaprotein genes [pPFT1Csf-3t(1)] and the cry1C-t transcript lev-

FIG. 2. Transcript levels for different cry1C-t constructs. Lane 1, pPFT1C-t(cyt1A p 1 cry1C-t); lane 2, pPFT1Cs-t (cyt1A p 1 STAB-SD 1 cry1C-t); lane3, pPFT1Cs-3t(1) (cyt1A p 1 STAB-SD 1 cry1C-t 1 stem-loop [59-39]); lane 4,pPFT1Cs-3t(2) (cyt1A p 1 STAB-SD 1 cry1C-t 1 stem-loop [39-59]); lane 5,pPFT1Csf-3t(1) (cyt1A p 1 STAB-SD 1 orf2 1 cry1C-t 1 stem-loop [59-39]);lane 6, pPFT1Cs-20k (cyt1A p 1 STAB-SD 1 cry1C-t 1 20-kDa protein gene);lane 7, pPFT1Csf-20k (cyt1A p 1 STAB-SD 1 orf2 1 cry1C-t 1 20-kDa proteingene). The ratios shown for lanes 2 through 7 are relative to the value for the dotin lane 1, which was assigned a value of 1. Each value represents the averagevalue (ratio) obtained from three separate experiments. Different letters beneaththe ratios indicate that values were significantly different at P 5 0.05.

TABLE 2. Primers used to amplify cry1C, cry2A, cry3A,and cry11A sequences

Primer Sequencea

1Ca-1 .........59-ACGCGTCGACCGGAGGTATTTTATGGAGGAAAAT-391Ca-2 .........59-ACGCGTCGACTTACTTTTGTGCTCTTTCTAAATCAGA-391Ca-3 .........59-ACATGCATGCCCCCTTAGATAGATATCATAGAATTG-393Aa-1.........59-ACATGCATGCATTAACTAGAAAGTAAAGAAGTAG-393Aa-2.........59-ACATGCATGCAAGCTTACAGAGAAATACACGAGGG-392Aa-1.........59-GCTCTAGAATAGGAGGAAAAGATTTTATGCTAAAA-392Aa-2.........59-ACGCGTCGACAAATATCTAGTTTTATATTAA-3911Aa-1.......59-ACATGCATGCAGTCATGTTAGCACAAGAGGA-3911Aa-2.......59-ACATGCATGCTTTAGGTCTTTAAAAATTAGA-39

a The ribosome binding site, start codon, and artificial stop codon are indicatedby boldface type, and the SalI, SphI, and XbaI restriction enzyme sites areunderlined.

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els with constructs that contained the 20-kDa protein genes(pPFT1Cs-20k) (Fig. 2, lanes 5 and 6, respectively). These lev-els were only 62 and 66%, respectively, of those detected withpPFT1Cs-3t(1). However, inclusion of the genes encoding boththe 29- and 20-kDa proteins in a construct (pPFT1Csf-20k)(Fig. 1; Table 1) increased the cry1C-t transcript level to a lev-el comparable to that observed with pPFT1Cs-3t(1) (Fig. 2,lanes 3 and 7).

Synthesis of Cry1C-t and inclusion formation. Constructswhich lacked the STAB-SD and cry3A 39 stem-loop sequences(pPFT1C-t) (Fig. 1) or contained the STAB-SD sequence andcry3A stem-loop in the orientation opposite that of cry1C-t[pPFT1Cs-3t(2)] produced little or no detectable Cry1C-t 68-kDa protein as determined by Western blot analysis (Fig. 3Aand B, lanes 1 and 4). No Cry1C-t inclusions were observed in

strains transformed with these constructs (Fig. 4A). However,when various elements that stabilized the transcript or en-hanced net protein synthesis were included in the constructs,substantial increases in Cry1C-t levels were obtained, and in-clusions of this protein were observed (Fig. 3A and B, lanes 2,3, 5, 6, and 7, and 4B and C). For example, when the STAB-SDsequence was included in pPFT1C-t (pPFT1Cs-t) (Fig. 1; Ta-ble 1) or the cry3A stem-loop sequence, placed in the sameorientation as cry1C-t, was included in pPFT1Cs-t [pPFT1Cs-3t(1)] (Fig. 1; Table 1), the constructs produced Cry1C-t (Fig.3A and B, lanes 2 and 3). Comparable levels of Cry1C-t werealso detected with constructs that contained the 29- or 20-kDaprotein genes (Fig. 3A and B, lanes 5 and 6), though inclusionswere observed in only about 33% of the sporulating cells (datanot shown). The sizes of these inclusions were similar to those

FIG. 3. Synthesis of Cry1C-t by different constructs as determined by SDS-PAGE and Western blot analysis. (A and B) SDS–12% PAGE gel (A) and Western blotof the same gel (B). The relative amounts of Cry1C-t produced by the strains are indicated below the lanes in panel A. Lane 1, pPFT1C-t (cyt1A p 1 cry1C-t); lane2, pPFT1Cs-t (cyt1A p 1 STAB-SD 1 cry1C-t); lane 3, pPFT1Cs-3t(1) (cyt1A p 1 STAB-SD 1 cry1C-t 1 stem-loop [59-39]); lane 4, pPFT1Cs-3t(2) (cyt1A p 1 STAB-SD 1 cry1C-t 1 stem-loop [39-59]); lane 5, pPFT1Csf-3t(1) (cyt1A p 1 STAB-SD 1 orf2 1 cry1C-t 1 stem-loop [59-39]); lane 6, pPFT1Cs-20k (cyt1A p 1 STAB-SD 1cry1C-t 1 20-kDa protein gene); lane 7, pPFT1Csf-20k (cyt1A p 1 STAB-SD 1 orf2 1 cry1C-t 1 20-kDa protein gene); lane M, molecular size marker. (C) Controlfor the Western blot analysis: B. thuringiensis subsp. israelensis, which produces Cry11A and Cyt1A. Lane 1, SDS-PAGE gel; lane 2, Western blot of the same gel. Theratios shown for lanes 3 through 7 are relative to the amount of the 58-kDa protein in lane 2, which was assigned a value of 1. Each value represents the average value(ratio) obtained from three separate experiments. Different letters beneath the ratios indicate that values were significantly different at P 5 0.05.

FIG. 4. Phase-contrast micrographs of sporulated cells of B. thuringiensis subsp. israelensis 4Q7 that expressed representative constructs. (A) pPFT1C-t (cyt1A p 1cry1C-t); (B) pPFT1Cs-3t(1) (cyt1A p 1 STAB-SD 1 cry1C-t 1 stem-loop [59-39]); (C) pPFT1Csf-20k (cyt1A p 1 STAB-SD 1 orf2 1 cry1C-t 1 20-kDa protein gene).The arrows indicate inclusions formed by Cry1C-t.

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in cells with pPFT1Cs-3t(1). The inclusions produced by theconstruct containing both the 20- and 29-kDa protein genes(pPFT1Csf-20k) (Fig. 1; Table 1) appeared to be larger thanthose produced by the other constructs (Fig. 4C). In most cells,two separate inclusions were observed in each cell when theconstructs contained either the 20-kDa (pPFT1Cs-20k), 29-kDa [pPFT1Csf-3t(1)], or 20- and 29-kDa (pPFT1Csf-20k)protein genes (Fig. 4C).

A putative 68-kDa band corresponding to Cry1C-t was notobserved in protein profiles of strains containing pPFT1Cs-t,pPFT1Cs-3t(1), pPFT1Csf-3t(1), pPFT1Cs-20k, and pPFT1Csf-20k, although at least three novel proteins with molecularmasses ranging from 55 to 58 kDa were observed (Fig. 3A,lanes 2, 3, 5, 6, and 7, respectively). Inclusions that were spher-ical or ovoidal were present in cells transformed with each ofthese plasmids. The level of Cry1C-t synthesis (Fig. 3A and B)generally corresponded with the cry1C-t transcript levels de-tected (Fig. 2). For example, constructs which contained theSTAB-SD and cry3A stem-loop sequences in the same orien-tation as cry1C-t [pPFT1Cs-3t(1)] and constructs that con-tained the 29- and the 20-kDa protein genes along with theSTAB-SD sequence (pPFT1Csf-20k) showed the highest levelsof cry1C-t transcript and subsequent synthesis of the corre-sponding protein. The protein yields of the strains containingthese constructs were, respectively, 1.4- and 1.7-fold greaterthan the amount of Cry1C-t produced by pPFT1Cs-t, whichlacked the cry3A stem-loop sequence and helper protein genes(i.e., the genes encoding the 29- and 20-kDa proteins) (Fig. 2and 3B, lanes 3 and 7). Similar levels of transcript and Cry1C-tsynthesis occurred in strains transformed with constructs con-taining either the 29-kDa protein gene [pPFT1Csf-3t(1)] orthe 20-kDa protein gene (pPFT1Cs-20k) (Fig. 2 and 3, lanes 5and 6). These strains produced, respectively, 1.2- and 1.3-foldmore Cry1C-t than the strain with pPFT1Cs-t produced.

Although the masses of the 55- to 58-kDa proteins did notcorrespond to the predicted mass of Cry1C-t (68 kDa), West-ern blotting with anti-Cry1C antibody confirmed that theirbands were composed of Cry1C-t (Fig. 3B, lanes 2, 3, 5, 6,and 7). SDS-PAGE and Western blot analyses of purifiedinclusions showed that the major component was the 68-kDaCry1C-t molecule (Fig. 5). Together, these results indicate thatthere is considerable degradation of Cry1C-t after synthesis(Fig. 3B and 5B).

Toxicity of Cry1C-t. The results of bioassays performed withneonate S. exigua larvae are shown in Tables 3 and 4. Twoconstructs that had the highest levels of Cry1C-t synthesis werecompared with the construct containing full-length cry1C byusing B. thuringiensis subsp. israelensis 4Q7 as the host strain ineach case (Table 3). Neither Cry1C-t construct was nearly astoxic as the full-length molecule. The 50% lethal concentra-tions (LC50s) for pPFT1Cs-3t(1), which contained the STAB-SD and cry3A stem-loop sequences in the same orientationas cry1C-t, and pPFT1Csf-20k, which contained STAB-SD plusthe 29- and 20-kDa protein genes, were 23.4 and 13.2 mg/ml,respectively, whereas pPF1C, which included the cry1C pro-moter to drive full-length cry1C, was significantly more toxic(LC50 5 1.9 mg/ml).

Bioassays were also performed with the HD1 isolate ofB. thuringiensis subsp. kurstaki (from Dipel; Abbott Laborato-ries) and B. thuringiensis subsp. aizawai 1857 (Xentari; AbbottLaboratories) transformed with cry1C-t and full-length cry1Cconstructs (Table 4). For cry1C-t, the construct yielding thehighest level of truncated Cry1C, pPFT1Csf-20k, was used, andfor full-length cry1C, pPF1C was used. The LC50s of HD-1/pPFT1Csf-20k and HD-1/pPFT1Cs preparations were 7.3 and3.5 mg/ml, respectively, demonstrating that both strains weremore toxic to S. exigua larvae than was the wild-type strainB. thuringiensis subsp. kurstaki HD-1, which had an LC50 of17.6 mg/ml. However, no significant differences were ob-served in the LC50s of preparations of B. thuringiensis subsp.aizawai 1857 and the strains harboring cry1C-t (1857/pPFT1Csf-20k) or cry1C (1857/pPFT1Cs).

DISCUSSION

cry1C-t was expressed in this study by using the cyt1A pro-moter–STAB-SD system (13) in combination with various oth-er genetic elements shown previously to enhance Cry proteinsynthesis. These included the cry3A transcription terminator(18) and the 20- and 29-kDa so-called helper proteins thatimprove net synthesis and crystallization. The highest yieldof Cry1C-t was obtained with a construct that included the 59

FIG. 5. Purified inclusions of Cry1C-t as determined by SDS-PAGE andWestern blot analysis. (A) SDS–10% PAGE gel; (B) Western blot of the samegel. Lane 1, molecular size marker; lanes 2 and 3, pPFT1Cs-3t(1) (cyt1A p 1STAB-SD 1 cry1C-t 1 stem-loop [59-39]); lanes 4 and 5, pPFT1Csf-20k (cyt1Ap 1 STAB-SD 1 orf2 1 cry1C-t 1 20-kDa protein gene). Five micrograms (lanes3 and 5) or 10 mg (lanes 2 and 4) of protein was loaded and separated.

TABLE 3. Comparative toxicities to neonate S. exigua of spore andinclusion mixtures of B. thuringiensis strains containing

full-length and truncated cry1C constructs

Straina LC50 (mg/ml) Slope 6 SEM

4Q7/pPF1C 1.9 (0.2–4.8)b 0.87 6 0.254Q7/pPFT1Cs-3t(1) 23.4 (13.5–37.5) 1.31 6 0.224Q7/pPFT1Csf-20k 13.2 (6.5–22.8) 1.22 6 0.21

a All strains were grown in 50 ml of NBG for 5 days at 30°C and lyophilized.b The values in parentheses are the 95% fiducial limits.

TABLE 4. Comparative toxicities to neonate S. exigua of spore andinclusion mixtures from commercial B. thuringiensis strains andstrains transformed with full-length and truncated cry1C genes

Straina LC50 (mg/ml) Slope 6 SEM

HD-1 (Dipel) 17.6 (9.7–30.5)b 1.53 6 0.30HD-1/pPFT1Csf-20k 7.3 (1.1–15.2) 1.17 6 0.36HD-1/pPFT1Cs 3.5 (1.8–7.4) 0.92 6 0.181857 (Xentari) 2.0 (1.1–3.3) 1.44 6 0.241857/pPFT1Csf-20k 2.0 (1.1–3.5) 1.19 6 0.211857/pPFT1Cs 1.6 (0.8–3.1) 0.99 6 0.19

a All strains were grown in 50 ml of NBG for 5 days at 30°C and lyophilized.b The values in parentheses are 95% fiducial limits.

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STAB-SD sequence, the 39 stem-loop structure, and genes en-coding the 20- and 29-kDa helper proteins. Synthesis of Cry1C-t with this construct also resulted in inclusions of Cry1C-tcrystals that were 50 to 80% the size of crystals formed by thefull-length molecule. These results suggest that use of these en-hancer elements in a single expression system might be usefulfor improving the synthesis of other truncated Cry1 proteins.

Previously, it was shown that the cry1Aa transcription termi-nator in either orientation at the distal end of the penicillinase(penP) gene or interleukin (IL-2) gene increased the half-lifeof their mRNAs by 2 to 6 min in Bacillus subtilis and Esche-richia coli (24). This resulted in a concomitant increase inPenP and IL-2 synthesis. In contrast to this, in the presentstudy a marked decrease in cry1C-t mRNA resulted when theorientation of the terminator was reversed (Fig. 2). This sug-gests that variations in cry transcription terminators may beone of the factors that determine the level of Cry synthesis.For example, Cry1C-t inclusions were not obtained withpPFT1C-t and pPFT1Cs-3t(2), constructs which lacked thestem-loop structure and contained the stem-loop structure in a39-59 orientation rather than a 59-39 orientation, respectively.Alternatively, Cry1C-t inclusions were obtained with all con-structs containing the stem-loop in the same orientation as itoccurs naturally in cry3A (18). The lack of synthesis detectedwhen the stem-loop was in the 39-59 orientation may have beendue to instability of the transcript.

The potential role of the cry11A operon 20-kDa and cry2Aoperon 29-kDa proteins on the level of synthesis and crystal-lization of truncated Cry1 proteins has received only limitedstudy. Rang et al. (15) showed that the 20-kDa protein in-creased net synthesis of Cry1C with deletions in the N-terminalhalf of the molecule but the proteins contained the C-ter-minal half of the molecule. Nevertheless, crystalline inclu-sions of these truncated proteins were not obtained. Simi-larly, the 20-kDa protein increased net synthesis of Cry2A butdid not directly enhance crystallization of this toxin in theabsence of the 29-kDa protein (7). In the present study, weshowed that when the 20- and 29-kDa protein genes were usedindependently, though the cry1C-t transcript levels were lowerthan the levels obtained when these genes were absent (Fig. 2),similar levels of Cry1C-t were produced (Fig. 3). This suggeststhat the 20- and 29-kDa proteins play no role in cry1C-texpression or mRNA stability but function to enhance netsynthesis of Cry1C-t.

The highest transcript level was obtained when both the 20-and 29-kDa protein genes were included in the construct.Thus, it appears that during transcription, cry1C-t was pro-tected from 59-39 and 39-59 exoribonuclease attack by STAB-SD–29-kDa protein and/or the 20-kDa protein gene fragmentsthat contained 59 and 39 stem-loop structures. It is likely thatthe increase in transcript stability contributed directly to thenet increase in the yield of Cry1C-t in cells with pPFT1Csf-20kby about 21% (Fig. 3, lane 7). Moreover, the presence of largeinclusions of Cry1C-t synthesized in cells with constructs con-taining the 20- and 29-kDa protein genes provides furtherevidence that the helper proteins which these genes encodecan function as molecular chaperones, promoting inclusionformation by Cry proteins other than Cry11A and Cry2A.

In previous studies it has been shown that truncated versionsof Cry1 proteins are unstable and have little or no toxicitycompared to the toxicity of full-length toxins (1, 15, 19, 21).The instability is probably due to the inability of these proteinsto fold and crystallize properly. For example, whereas the 20-kDa protein increased net synthesis of truncated Cry1C withdeletions in the N-terminal half, no crystalline inclusion wereobserved (15). This suggests that residues in the N-terminal

half of the molecule are essential for crystal formation. Here itis shown that Cry1C-t, which lacked the entire C terminus, wasable to form crystalline inclusions, even though these inclu-sions were approximately 50 to 80% the size of those producedby wild-type Cry1C. In addition, the SDS-PAGE and Westernblot data (Fig. 3) showed that Cry1C-t is unstable and is de-graded to smaller peptides with molecular masses ranging from55 to 58 kDa. There was a difference between the molecularmass of Cry1C-t molecules contained in purified crystallineinclusions (68 kDa) and the molecular masses of molecules inthe spore-crystal mixture (55 to 58 kDa). This probably re-sulted from degradation of the 68-kDa Cry1C-t molecules thatwere not occluded in the inclusions, as these would have beenexposed to proteases upon cell lysis (Fig. 5). The smaller 38-kDa peptides (Fig. 5), which were degraded to 31-kDa pep-tides (Fig. 3B), were possibly the translation products resultingfrom a second in-frame ATG codon, as noted previously (19).

It has been reported previously that Cry1C-t inclusions pro-duced in E. coli were toxic to Spodoptera littoralis (19). How-ever, this report contained no data on the relative toxicity ofCry1C-t or full-length Cry1C molecules to S. littoralis. Thesequence of the Cry1C-t used in the present study was identicalto the sequence of the Cry1C-t described previously (19).Here, it is shown that Cry1C-t produced in B. thuringiensis isalso toxic to larvae of the beet armyworm, S. exigua, althoughthe toxicity of the truncated protein was 7- to 12.6-fold lessthan that of the full-length Cry1C (Table 3). The reasons forthe lower toxicity of the truncated form are not known atpresent, but the difference could be due to lower stability andperhaps incorrect folding in the absence of the large C termi-nus. Despite the lower toxicity of Cry1C-t, synthesis of thismolecule in the HD1 isolate of B. thuringiensis subsp. kurstaki,the isolate used in the commercial insecticide Dipel, whichlacks Cry1C, increased toxicity to S. exigua as much as fivefold(Table 4). This indicates that the N-terminal half of Cry1C wassynthesized effectively in this strain, a finding that may increasethis strain’s commercial utility. A similar result was not ob-served with B. thuringiensis subsp. aizawai (Table 3), most like-ly because this strain already contains Cry1C.

ACKNOWLEDGMENTS

We thank Jeffrey J. Johnson for assistance during the course of thisstudy.

This research was supported in part by grant 96-51 to B.A.F. fromthe University of California BioSTAR Program.

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