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Applied and Environmental Microbiology, August 2008, p. 5178-5182, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00598-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Production of an Insecticidal Crystal Protein from Bacillus thuringiensis by the Methylotroph Methylobacterium extorquens

Young J. Choi,¹ J. Lawrence Gringorten,² Louise Bélanger,¹ Lyne Morel,¹ Denis Bourque,¹ Luke Masson,^1,3 Denis Groleau,¹ and Carlos B. Míguez^1*

Microbial and Enzymatic Technology Group, Bioprocess Sector,¹ Environmental Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada,³ Canadian Forest Service, Great Lakes Forestry Centre, 1219 Queen Street E., Sault Ste. Marie, Ontario P6A 2E5, Canada²

Received 12 March 2008/ Accepted 9 June 2008

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The Cry1Aa protein from Bacillus thuringiensis is an insecticidal protein that is highly active against several species of Lepidoptera. We cloned and expressed the cry1Aa gene in a plant-colonizing methylotroph, Methylobacterium extorquens, under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter, P_mxaF. Transmission electron microscopy revealed characteristic bipyramidal intracellular -endotoxin crystals similar to the crystalline inclusions formed by B. thuringiensis. Both the protoxin protein and the activated toxin were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis. In single-dose assays of the recombinant against the silkworm, Bombyx mori, both whole cells and cell lysates caused rapid feeding inhibition followed by mortality. The biomass and growth rate of recombinant cells in shake flask culture were similar to those of the wild-type strain, indicating a lack of fitness cost to the recombinant under controlled culture conditions. Recombinant Cry1Aa was expressed at a level of 4.5% of total M. extorquens cell protein. The potential benefits of modifying M. extorquens to deliver insecticidal Cry proteins for crop and forest protection are discussed.

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Methylotrophs comprise a diverse group of bacteria that grow on single carbon substrates, e.g., methane or methanol, as the sole source of organic carbon and energy. They are ubiquitous in nature, in a variety of epi- and endophytic associations with plants and trees, and have been isolated from almost all leaf surfaces tested (16, 20, 30, 32, 40). Methylobacterium extorquens is one of several pink-pigmented facultative methylotrophs (PPFMs) that colonize plants and utilize the methanol produced by leaves as a by-product of cell wall synthesis (15, 35). Certain PPFMs are known phytosymbionts (10, 23, 36). M. extorquens has been described as an endophyte in the stems and leaves of citrus plants (24) and a bud endophyte and symbiont of Scots pine (31), as well as a colonizer of several other tree species (11).

The availability of genetic tools (8, 9, 13, 28), sequenced genomes, and stoichiometric models for evaluating its metabolic capabilities (37) has stimulated economic interest in M. extorquens as a production host for various bioproducts. Heterologous expression of several bacterial proteins in M. extorquens has been previously reported (3, 8, 9, 13, 14, 18).

In this study, we cloned the cry1Aa -endotoxin gene from Bacillus thuringiensis subsp. kurstaki under the control of the P_mxaF promoter in M. extorquens ATCC 55366. The cry1 gene class encodes potent insecticidal proteins in the form of crystalline protoxins of 120 to 157 kDa (22). They are particularly active against larvae of lepidopteran defoliators, including the spruce budworm, Choristoneura fumiferana, and gypsy moth, Lymantria dispar (38), two highly destructive North American forest insects. In the larva, upon ingestion, protoxins are proteolytically cleaved by midgut enzymes, releasing active toxins of 60 to 70 kDa (12). Activated toxins bind to and destroy the midgut epithelium of susceptible insects.

Although ubiquitous in nature and found in the phylloplane (4, 7, 27, 34), B. thuringiensis, unlike Methylobacterium sp., does not normally colonize foliage and propagate vegetatively (25). When sprayed as an insecticidal agent, its persistence on foliage is relatively short due to UV inactivation, heat, and rain washoff (17). The intimate association between M. extorquens and host plants suggests that a novel use of this organism would be to modify it to deliver insecticidal proteins for plant protection. In the present report, we describe the first stage of this work involving the cloning and expression of the cry1Aa gene in M. extorquens and confirm insecticidal activity. This is the first study to examine the use of M. extorquens as a host for recombinant insecticide production.

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Bacterial strains, plasmids, and growth conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. M. extorquens was grown in 250 ml Choi 4 medium in shake flasks, as previously described (3, 5), with 0.5% (vol/vol) methanol as the sole carbon source. Escherichia coli was cultured in Luria-Bertani broth at 37°C. Recombinant cells harboring the pCry1Aa plasmid were grown in medium that also contained 20 µg/ml tetracycline. Both media were solidified with 1.8% agar (Difco) when required. M. extorquens cultures were grown at 30°C to the late logarithmic phase (48 h), harvested, washed, and resuspended in phosphate-buffered saline (PBS) (2.7 mM KCl, 10 mM PO₄, 137 mM NaCl, pH 7.4). Cell densities and biomass yields were determined from optical density readings at 600 nm referenced to density and dry-weight calibration curves, respectively.

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TABLE 1. Organisms and plasmids used in this study

DNA isolation and manipulations.
Plasmids from E. coli were prepared with the Qiagen mini-plasmid purification kit according to the manufacturer's instructions (Qiagen Inc., Mississauga, ON, Canada). Basic molecular biology techniques were implemented as previously described (33). DNA fragments were isolated from agarose gels with the Qiaquick gel extraction kit (Qiagen). T4 DNA ligase and other DNA-modifying enzymes were purchased from New England Biolabs, Inc.; Gibco/BRL Life Technologies, Inc.; or Pharmacia LKB Biotechnology and used as recommended by the manufacturers. Electroporation was performed with a Gene-Pulser II electroporation apparatus (Bio-Rad Laboratories, Mississauga, ON, Canada).

Plasmid construction.
The expression plasmid pMP39 containing the cry1Aa gene from B. thuringiensis subsp. kurstaki strain NRD-12 (29) was used as a PCR template. The DNA sequence encoding the Cry1Aa protein and ribosome binding site in pMP39 was amplified using primers BTF80 (5'-CGG ATC CAT GGA TAA CAA TCC GAA CAT CAA TG –3') and BTR80 (5'-CGG ATC CCT ATT CCT CCA ATA AGG AGT AAT TC-3') (BamHI restriction enzyme sites are underlined). The PCR product was then cloned into the pCR2.1-TOPO vector, generating pCR-cry1Aa. From the latter plasmid, the gene was cloned into the broad-host-range vector pCM80 (28), digested with BamHI, and dephosphorylated, forming pCry1Aa (Fig. 1). To confirm the orientation of the cry1Aa fragment in the vector, plasmid DNA was extracted from clones and analyzed by restriction mapping and sequencing. Following confirmation, pCry1Aa was transformed in M. extorquens by electroporation. A second construct, pRCry1Aa, in which the cry1Aa gene was inserted in reverse orientation to the P_mxaF promoter, was also cloned into M. extorquens and used as a negative control.

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FIG. 1. Construction of recombinant plasmid pCry1Aa.

Transmission electron microscopy.
Cells from liquid cultures were collected, pelleted, and fixed in 4% paraformaldehyde, 2.5% glutaraldehyde (100 mM sodium cacodylate buffer, pH 7.3) for 24 h at 4°C. After postfixation in 1% OsO₄ (in the same buffer) for 90 min at 20°C, the samples were dehydrated in an ethanol/propylene oxide series and embedded in Epon. Ultrathin sections of 80 nm were produced with a Reichert-Jung Ultramicrotome (Vienna, Austria), transferred to a 200-mesh nickel/Formvar grid, and stained with 3% uranyl acetate/lead citrate. Samples were examined in a transmission electron microscope (JEM-1230; Jeol, Tokyo, Japan) at 80 kV.

Preparation of cell lysates.
Recombinant M. extorquens cells suspended in PBS were lysed by three passes through a French pressure cell at 76 MPa internal pressure. The material was diluted and resuspended in sterile deionized water, assayed for insecticidal activity, and processed further for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and determination of the Cry1Aa protein yield. Endotoxin crystals, as well as other insoluble material, were recovered in the precipitate following centrifugation of the lysate suspension at 10⁴ x g and 4°C for 20 min. For immunological identification of Cry1Aa proteins by Western analysis, lysates were also prepared in which the cells were first resuspended in a urea lysis buffer (50 mM Tris, 50 mM EDTA, 8 M Urea, pH 8.0), thereby solubilizing the crystal inclusions upon French press treatment. In this case, endotoxin protein was recovered in the supernatant following centrifugation.

Detection of Cry1Aa recombinant protein by SDS-PAGE.
Protoxin and activated toxin were revealed by SDS-PAGE of alkaline extracts and enzymatic digestions of the insoluble fraction of PBS lysates. The lysate suspension was centrifuged, and the precipitate was cycled through three washes in 0.01% Triton X-100 in 1 M NaCl, followed by three more washes and resuspension in sterile deionized water. For protoxin analysis, the washed lysate was extracted at 28°C for 2 h in 0.1 M CAPS (N-cyclohexyl-3-aminopropanesulfonic acid)-KOH buffer, pH 10.5 (pH 10.5 buffer), containing 0.2% dithiothreitol, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and 5 mM o-phenanthroline; clarified by centrifugation; and dialyzed in 50 kDa MWCO tubing (SpectraPor 6) against pH 10.5 buffer. For activated toxin, the lysate was digested in 1% B. mori gut juice in pH 10.5 buffer at 28°C for 20 h, followed by centrifugation. Further digestion was stopped by the addition of 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride and o-phenanthroline to the supernatant to final concentrations of 1 mM and 5 mM, respectively. Protein concentrations of alkaline buffer extracts and gut juice digests were determined by the method of Bradford (6), using the Bio-Rad microassay protein procedure and bovine serum globulin as a standard. The electrophoresis was carried out on 12% gels, and proteins were visualized with Coomassie brilliant blue R-250. The gels were analyzed by scanning densitometry to estimate the recombinant protein yield (below).

Immunological identification of Cry1Aa protein.
The urea lysate was analyzed by SDS-PAGE using NuPage 4 to 12% Bis-Tris gels (Invitrogen), and unstained gels were electroblotted onto nitrocellulose. Recombinant Cry1Aa was identified using rabbit anti-Cry1Aa protoxin polyclonal antibodies. Detection was carried out using goat anti-rabbit immunoglobulin G (H+L) labeled with horseradish peroxidase (Pierce Inc., West Grove, PA).

Biological assays.
Single-dose assays of the recombinant were performed against fourth-instar silkworms, B. mori, to verify the presence of insecticidal activity. Whole-cell and PBS-lysate samples were diluted in sterile deionized water to a protein concentration of 24 mg/ml, and 6 µl was force fed to day 1 fasting larvae, using an electronic microinjector system. Controls were divided into three groups: (i) larvae force fed pRCry1Aa recombinant, (ii) larvae force fed water only, and (iii) untreated larvae. Following treatment, the insects were placed in petri dishes with mulberry leaves. Feeding inhibition was qualitatively assessed by the relative amount of frass produced, and mortality was scored after 48 h.

Recombinant protein yield.
The relative yield of Cry1Aa was determined from lysed samples as the percentage of protoxin relative to total cell protein. The absolute yield was measured as the amount (in femtograms) produced per cell. Quantification of the protoxin and determination of molecular mass were carried out by scanning densitometry of stained SDS-PAGE gels at 595 nm. Due to the uncertainty of full recovery of the protoxin protein (due to possible losses from partial breakdown in alkaline extracts and during dialysis), protoxin was quantified indirectly from the value obtained for activated toxin multiplied by the protoxin/toxin M_r ratio. Protoxin and total protein concentrations were normalized to mg/ml values of lysate suspension, and their ratio was used to estimate the relative yield (mg/mg) times 100%. The absolute yield of protoxin (fg/cell) was similarly determined from the known density of the cell suspension that was lysed and normalized to cells/ml lysate suspension.

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Transmission electron microscope images showed that recombinant M. extorquens produced intracellular crystal-shaped inclusions (Fig. 2) similar to those produced by sporulating cells of B. thuringiensis subsp. kurstaki (39). They were typically bipyramidal in shape and measured approximately 0.25 µm in length, about one-quarter the size of B. thuringiensis subsp. kurstaki crystals. SDS-PAGE of PBS lysates revealed protoxin and activated-toxin bands at 137 kDa and 60 kDa, respectively (Fig. 3). The M_r ratio 137/60 was used as the conversion factor to calculate the protoxin amount from activated toxin and to estimate the yield of recombinant protein. Immunoblots of urea lysates confirmed the presence of protoxin, activated toxin, and, especially in the case of the NRD-12 control, a protein fraction of intermediate M_r representing partially digested protoxin (Fig. 4). The presence of activated toxin and intermediate bands was attributed to endogenous protease activity during protoxin synthesis and crystal formation.

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FIG. 2. Transmission electron micrographs of wild-type (A) and Cry recombinant (B and C) M. extorquens. The arrows indicate -endotoxin crystals, and the bars represent 0.2 µm (A and C) and 0.5 µm (B).

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FIG. 3. SDS-PAGE of PBS-buffered lysed cells of Cry (active) and RCry (control) recombinant M. extorquens. Lanes: 1, supernatant from Cry recombinant; 2, KOH extract of precipitate from RCry recombinant; 3, KOH extract of precipitate from Cry recombinant; 4, gut juice digest of precipitate from RCry recombinant; 5, gut juice digest of precipitate from Cry recombinant; 6, molecular mass markers. Arrowheads: lane 1, methanol dehydrogenase; lane 3, Cry1Aa protoxin; lane 5, Cry1Aa activated toxin.

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FIG. 4. Immunoblot of Cry1Aa protoxin (P) and activated toxin (T) in the supernatant of urea-buffered lysed cells of Cry recombinant M. extorquens. Lanes: 1, M. extorquens wild-type control; 2 and 3, Cry recombinant (1 µg protein); 4, B. thuringiensis subsp. kurstaki strain NRD-12 (0.1 µg protein).

Recombinant M. extorquens bioassay results against B. mori are summarized in Table 2. B. mori was used as the test insect due to its relatively large size and very high susceptibility to Cry1Aa. In the experimental groups, both whole cells and lysates of the recombinant caused rapid feeding inhibition and 100% mortality. None of the control groups displayed comparable feeding inhibition.

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TABLE 2. Response of B. mori larvae force fed recombinant M. extorquensa

The presence of intracellular crystalline inclusion bodies, the detection and identification of protoxin and activated toxin proteins by SDS-PAGE and Western analysis, and the demonstration of insecticidal activity confirmed that we were able to clone the cry1Aa gene into M. extorquens and control its expression constitutively using the P_mxaF promoter. To our knowledge, Methylobacillus flagellatum is the only other methylotroph engineered with an insecticidal protein. It expressed the mosquitocidal cry4B gene from B. thuringiensis subsp. israelensis under the control of a lac promoter (26). Comparisons of growth rate, biomass, and total protein production between Cry, RCry, and wild-type cells (Table 3) showed that the growth profile of M. extorquens cells was unaffected by recombinant protein production. These results are consistent with an earlier study of green fluorescent protein expression in M. extorquens (3).

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TABLE 3. Growth profiles of wild-type and Cry1Aa recombinant M. extorquens in shake flask cultures

Quantitative analysis of the recombinant protein produced values of 4.5% of total protein and 9.8 fg/cell. Combining the latter figure with the known density of the culture when the cells were harvested (5 x 10⁸ cells/ml after 48 h), the average rate of protoxin synthesis, under the present culture conditions, was calculated to be approximately 0.1 µg/ml/h. Expression was low compared with the production of other heterologous proteins in M. extorquens and other microorganisms. At 4.5% of total cell protein, it is substantially lower than the yield reported for green fluorescent protein (16%), also cloned in M. extorquens and controlled by the P_mxaF promoter (3). Barnes and Cummings (2) cloned the cry1Ac gene with its native promoter in a closely related, but nonmethylotrophic, leaf-colonizing bacterium, Pseudomonas fluorescens, and achieved expression levels of 10 to 20% of total cell protein (17). Armengol et al. (1) cloned the mosquitocidal cry11Aa gene with the tac promoter in the aquatic bacterium Asticcacaulis excentricus and reported a yield of 0.04 pg recombinant protein per cell, about four times the amount of Cry1Aa obtained in this study. The reason for the lower-than-expected yield of Cry1Aa in M. extorquens is a matter of speculation, but it may be related to either protein or mRNA stability. Although crystal formation was a prominent feature of the recombinant cells, the proportion of total crystal protein consisting of protoxin is not known.

Ultimately, any practical application of a modified M. extorquens strain expressing Cry toxin at a lower level than commercial B. thuringiensis products would have to rely on other features, such as persistence or cost, that provide advantages which compensate for the lower yield. A case in point is the recombinant P. fluorescens expressing cry1Ac (2, 17). Field tests of the killed organism showed that it had two to three times the foliar persistence of B. thuringiensis (perhaps due to greater resistance to UV degradation), prompting its registration for use as a commercial biopesticide (17). An effort to improve the yield of Cry1Aa in M. extorquens, therefore, might be considered worthwhile.

From the standpoint of its ability to colonize plants as an epi- or endophyte, a modified M. extorquens strain would appear to be an ideal host to deliver B. thuringiensis insecticidal proteins for crop and forest protection. Internal density levels of Methylobacterium sp. in plant tissue can reach as high as 10⁸ CFU/g fresh weight (21). As a true colonizer actively metabolizing and proliferating in the phylloplane, the persistence of M. extorquens on foliage should be longer than that of B. thuringiensis. The latter, when present on foliage, normally exists as a dormant spore (25), susceptible to UV degradation and rain washoff. On the other hand, PPFMs cannot be entirely removed from plant material by washing or even surface sterilization (19, 21) but require phage treatment to achieve total elimination (19). In contrast to the exposed state of B. thuringiensis crystals following spray operations, M. extorquens recombinant protoxin would remain packaged as an intracellular inclusion within the cell, providing protection against UV effects and washoff. Field applications of insecticidal Cry proteins in recombinant M. extorquens, therefore, could have the benefit of longer residual activity than B. thuringiensis.

Natural epi- and endophytic associations of Methylobacterium sp. have been reported in several deciduous and coniferous trees, including pine, spruce, and maple (11), suggesting that a recombinant able to colonize host trees could deliver B. thuringiensis insecticidal proteins and provide superior protection against destructive forest insects. A high-cell-density fermentation system already exists for mass producing M. extorquens (3, 5), and the reliance on methanol as the sole organic substrate for growing it affords a relatively low-cost production system compared with the manufacturing costs for B. thuringiensis and other nonmethylotrophs.

To avoid recombinant plasmid instability under field conditions, we intend to integrate the cry1Aa gene into the chromosome of M. extorquens using a mini-Tn7 transposon system (9). In addition to stable gene expression, chromosomal integration will obviate the need for selection pressure (9). The results of cry1Aa integration and plant colonization will be reported in a later communication.

 
We thank Richard Janvier (Université Laval, Que., Canada) for his assistance with the electron microscope results and Anthony Pang (Great Lakes Forestry Centre, Ont., Canada) for generously providing the anti-protoxin antibodies.

 
* Corresponding author. Mailing address: Microbial and Enzymatic Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Phone: (514) 496-6280. Fax: (514) 496-7251. E-mail: carlos.miguez{at}nrc-cnrc.gc.ca

Published ahead of print on 13 June 2008.

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Applied and Environmental Microbiology, August 2008, p. 5178-5182, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00598-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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