MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 96(2) February 2001
PAGES: 257-263 DOI: Full paper
Expression of Mosquito Active Toxin Genes by a Colombian Native Strain of the Gram-negative Bacterium Asticcacaulis excentricus

Magally Romero, Flor M Gil, Sergio Orduz +

Unidad de Biotecnología y Control Biológico, Corporación para Investigaciones Biológicas, Apartado Aéreo 7378, Medellín, Colombia

Abstract

Mosquito control with biological insecticides, such as Bacillus sp. toxins, has been used widely in many countries. However, rapid sedimentation away from the mosquito larvae feeding zone causes a low residual effect. In order to overcome this problem, it has been proposed to clone the Bacillus toxin genes in aquatic bacteria which are able to live in the upper part of the water column. Two strains of Asticcacaulis excentricus were chosen to introduce the B. sphaericus binary toxin gene and B. thuringiensis subsp. medellin cry11Bb gene cloned in suitable vectors. In feeding experiments with these aquatic bacteria, it was shown that Culex quinquefasciatusAedes aegypti, and Anopheles albimanus larvae were able to survive on a diet based on this wild bacterium. A. excentricus recombinant strains were able to express both genes, but the recombinant strain expressing the B. sphaericus binary toxin was toxic to mosquito larvae. Crude protease A. excentricus extracts did not degrade the Cry11Bb toxin. The flotability studies indicated that the recombinant A. excentricus strains remained in the upper part of the water column longer than the wild type Bacillus strains.

Bacillus thuringiensis, a gram-positive bacterium produces toxins active against some insect species belonging to the orders Coleoptera, Diptera, and Lepidoptera. Several highly toxic strains of B. thuringiensis have been reported for mosquito control (Goldberg & Margalit 1977, Padua et al. 1984, Orduz et al. 1992, Seleena et al. 1995, Ragni et al. 1996). The classification of the mosquito active strains in three groups, proposed by Delécluse et al. (1995), positions strains potentially important for mosquito control in group 2. These include two strains of B. thuringiensis subspeciesmedellin, strain 367 of the subsp. jegathesan, and Clostridium bifermentans serovar. malaysia. These four strains display a crystal protein pattern different from that found in B. thuringiensis subsp. israelensis, while are nearly as active. Polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified parasporal crystalline inclusions from B. thuringiensis subsp. medellin strain CIB 163-131 shows a polypeptide of approximately 94 kDa, multiple bands between 80 and 65 kDa, and two doublets at 40-41 and 28-30 kDa. The sequence of the 94 kDa toxin gene of B. thuringiensissubsp. medellin encoding the Cry11Bb1 protein and its genetic organization has been reported (Orduz et al. 1998).

Parasporal inclusions containing the mos-quitocidal toxins rapidly settle on the bottom of the ponds, away from mosquito larvae feeding zone (Murphy & Stevens 1992, Thanabalu et al. 1992, Xudong et al. 1993, Orduz et al. 1995). In order to overcome this problem, it has been proposed to clone mosquito active toxin encoding genes in aquatic bacteria, such as gram negative and cyanobacteria. Several attempts have been done to clone B. thuringiensis subsp. israelensis toxin genes in cyanobacteria, obtaining variable results (Angsuthanasombat & Panyim 1989, Chungjatupornchai 1990, Murphy & Stevens 1992). At the same time, gram negative bacteria such as Asticcacalis excentricusCaulobacter crescentusand Ancylobacter aquaticus have been used to clone and to express B. thuringiensis subsp. israelensis toxin genes, providing better expression of gene products, and extended control by keeping toxins in larval environments at levels of 105 to 106 cells/ml (Thanabalu et al. 1992, Yap et al. 1994a,b).

In the present study we cloned the cry11Bb gene of B. thuringiensis subsp. medellin and the binary toxin gene of B. sphaericus in an A. excentricus strain isolated from mosquito larvae breeding ponds in Colombia, and present data on expression and toxicity of recombinant cells to A. albimanus, Ae. aegypti, and Cx. quinquefasciatus first instar larvae, as well as information regarding flotability properties of the recombinant strains.

 

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions - Strains JM109, and XL1 Blue MRF´ of Escherichia coli were obtained from Stratagene (La Jolla, CA, USA) and were grown at 200 rpm, 37oC in Luria-Bertani (LB) medium (Sambrook et al. 1989). Recombinant E. coli cells were grown at 200 rpm 37oC in LB medium supplemented with tetracycline 12 µg/ml. A. excentricus strain 4274 was obtained from the German Collection of Microorganism and Cell Cultures. Local A. excentricus strains were isolated from water samples taken from a lake near Medellin. A. excentricus cells were grown in PYE medium (2 g Bacto-peptone, 1 g yeast extract per liter), and their recombinants were grown in PYE supplemented with 6 µg of tetracycline per ml. B. sphaericus strain 2362 was grown in NYSM (g/l: nutrient broth, 28; yeast extract, 0.5 and supplemented with salt solution as follows: 0.7 mM CaCl2, 1 mM MgCl2, and 50 µM MnCl2) (Myers & Yousten 1978). Recombinant B. thuringiensis expressing the Cry11Bb protein was developed by Restrepo et al. (1997) and cultured by using M1 liquid medium. The Cry11Bb crystals were purified from a final whole culture (FWC) as described by Pendleton and Morrison (1966) with modifications introduced by Otieno-Ayayo et al. (1993). The FWC (250 ml) was washed twice in distilled water, the pellet resuspended in 0.5 M NaCl and gently stirred for 1 h at 30°C; after this, it was washed twice in distilled water supplemented with 0.1 M phenyl-methy-lsulfonyl-fluoride (PMSF) and centrifuged for 20 min at 12,000 rpm. The pellet was then added to a mixture of equal volumes of 1% Na2SO4 and CCl4. This mixture was vigorously shaken in a separation funnel for 10 min and allowed to sit until the formation of two layers. The aqueous phase containing crystals was washed three times in distilled water and stored at -20°C. Crystals were solubilized in 100 mM Caps, 0.05% b-mercaptoethanol, pH 10.6 for 1 h at 30°C, and insoluble material was removed by centrifugation at 12,000 rpm for 10 min. Protein concentration was determined by Bradford method (Bradford 1976) following the recommendations of the Protein Assay Kit (Bio-Rad).

Plasmid pBTM4 containing the cry11Bb1 gene was constructed by Restrepo et al. (1997). Plasmid pEA1 containing B. sphaericus binary toxin gene was a gift from Dr Alan Porter (Institute of Molecular and Cellular Biology, National University of Singapore). The B. sphaericus binary toxin gene cloned in plasmid pEA1 is under the control of the ptac1promoter, a synthetic sequence with the _35 and _10 regions of the tac promoter and a concensus ribosome binging site for C. crescentus, and downstream of the B. sphaericus binary toxin gene, a transcriptional terminator sequence from B.thuringiensis subsp. kurstaki cry gene (Yap et al. 1994a). Plasmid pSOD2 containing the cry11Bb gene of B. thuringiensis subsp. medellin was constructed as follows: the cry11Bb gene from plasmid pBTM4 was cloned into theEcoRI site of the plasmid pBlueScript SK(+) to give plasmid pDEMOND (6.3 kb). Plasmid pEA1 (12.4 kb) was digested with KpnI-PstI in order to release a 2.5 kb fragment containing the B. sphaericus binary toxin gene. An adapter with the sequence 5´-CTCGCGAAGCTTAGGCCTGCA-3´ was cloned in the KpnI-PstI sites of pEA1 adding the NruI-HindIII-StuI sites to give plasmid pSO1A. DNA sequence in both directions was determined in order to confirm the adapter sequence. pDEMOND was digested with HindIII, and fragment containing the cry11Bb gene (3.3 kb) was cloned into the HindIII site of plasmid pSO1A, producing plasmid pSOD2 (13.2 kb). Plasmids pEA1 and pSOD2 containing the same promoter and transcriptional terminator sequences were used to transform E. coli and A. excentricus cells.

Feeding experiments - Ae. aegypti and Cx. quinquefasciatus were collected in the vicinity of Medellin. A. albimanuscolony was established with several egg shipments sent by Dr Victor Olano (Colombian National Institute of Health). All mosquito colonies are maintained under laboratory conditions at 30±2°C under a 12:12 (light:dark) photoperiod. For the feeding experiments with all mosquito species, eggs were collected the same day, washed with double distilled and sterile water, and larvae were allowed to hatch. Ten larvae of each mosquito species were placed in sterile 50 mm diameter Petri dishes with 9 ml of sterile water. One ml of the Asticaccaulis strain C2 culture containing 1x107, 1x108 or 1x109 cells/ml was added. Asticcacaulis control without mosquito larvae was set to verify the viability of the suspensions. Mosquito larvae positive control treatments were also set with food (trout feed for Ae. aegypti and A.albimanus), and liver powder for Cx. quinquefasciatus. Growth of mosquito larvae and presence of exhuvii were recorded daily for five days. All treatments were set in triplicate and experiments were repeated in every two days. Every 24 h, and after gentle shaking of Petri dishes, 1 ml of suspension was withdrawn, and replaced with 1 ml of a fresh cell suspension in the Ae. aegypti and A. albimanus treatments, and every 48 h in Cx. quinquefasciatus treatments. Larvae of each mosquito species were set without food as negative control. Data were analyzed by Anova and means compared using Tukey´s test (Statistica, StatSoft, Inc.).

Transformation experiments - A. excentricus cells (strains 4724 and C2) were grown in PYE to an optical density of OD600 = 1.0, harvested by centrifugation at 7,000 rpm, 4oC, during 15 min, and washed three times alternatively with ice cold distilled water and ice cold 10% glycerol. Cells were finally resuspended in 200 µl 10% glycerol. Electroporation was used to transform 10 µl of A. excentricus cells with 3 µg of plasmids pSOD2 or pEA1. After the pulse, cells were placed in 1 ml of PYE and incubated for 2 h at 30oC. Recombinant cells were selected by plating in PYE agar containing 6 µg/ml of tetracycline and were grown for 48 h. Since no A. excentricus strain C2 recombinants were recovered, two alternative procedures were used to treat these bacterial strains: 20 successive passes on solid media, and treatment of the native strain with the ethyl ester of the methane sulfonic acid (EMS), as described by Tao et al. (1993). After treatments, cell suspensions were plated and colonies were checked to select those maintaining Asticcacaulismorphology to perform again the electrotransformation assays.

Electrophoresis and Western blot - Recombinant strains were analyzed by 10% SDS-PAGE and stained with Coomasie brilliant blue. Separated proteins were electrotransferred to nitrocellulose membranes, blocked overnight at room temperature in TBS (100 mM Tris-HCl, 150 mM NaCl, pH 7.2) with 3% gelatin, and washed three times for 5 min each in TBS with 0.05% Tween-20 (TTBS). Membranes were incubated for 1 h at room temperature with mouse anti-Cry11Bb or with anti-binary toxin polyclonal antibodies raised in mice, diluted 1:1000 in TTBS with 1% gelatin. After three washes in TTBS, membranes were incubated for 1 h with a goat anti-mouse IgG (H+L)-alkaline phosphatase conjugated (1:1000 in TTBS with 1% gelatin). After three washes, color was developed with a solution containing nitroblue tetrazolium chloride (1 mg/ml), 5-bromo-4-chloro-3-indolylphosphate (1.5 mg/ml), and 10 ml of 100 mM NaHCO3-Na2CO3, pH 9.86.

Mosquito larvicidal assays - Native and recombinant A. excentricus strains were grown in liquid PYE under shaking to an optical density of A600 = 1.0. B. thuringiensis subsp. medellin was grown in M1 at 30oC, 200 rpm, for 48 h until sporulation (Restrepo et al. 1997). Ae. aegypti and A. albimanus used in these bioassays are maintained under laboratory conditions as described by Restrepo et al. (1997). Petri dishes (50 mm diameter) were filled with 9.9 ml of distilled water, and 10 mosquito larvae of second instar were added to each dish. One hundred µl of serial dilutions of bacterial cultures were added to obtain dilutions ranging between 
10-2 and 10-7. Negative controls were set by adding 100 µl of distilled water. Each dose was performed by duplicate and repeated in three different days. One hundred µl of the bacterial cultures were plated on solid PYE or LB in order to determine bacterial concentration. Half lethal concentration (LC50) was determined by probit analysis.

Protease activity of A. excentricus extracts - Protease activity of A. excentricus extracts was tested in order to determine if Cry11Bb protein could be degraded in the cytoplasm of the recombinant bacteria. Cry11Bb protein was produced and purified as mentioned above. Five µg of toxin were incubated with 13 µg of crude protease extract prepared by sonication as described by Liu et al. (1996). Samples were ajusted to 30 µl with PBS buffer and incubated for 16 h at 30oC, separated by SDS-PAGE, and proteins were transferred to nitrocellulose membranes. Membrane was developed as mentioned before.

Flotation experiments - Cells of A. excentricus reference strain transformed and untransformed with plasmid pEA1,Asticcacaulis native strain C2, transformed with plasmid pSOD2 and untrans-formed, and final whole cultures of B.sphaericusB. thuringiensis subsp. medellin, B. thuringiensis subsps. israelensis and jegathesan were grown in appropriate media as indicated above, collected by centrifugation and resuspended to an A600 = 1 in water. Two ml were placed in sterile plastic cuvettes, sealed and kept at room temperature during six days. Cuvettes were photographed every two days in order to register their sedimentation.

 

RESULTS

Feeding experiments - Development of mosquito larvae was significatively different between controls (with and without normal food) and treatments containing A. excentricus cells as larval food in the three mosquito species tested; however, at the Aexcentricus lower concentrations (107 and 108 cells/ml) larvae reached only second instar after five days, while in treatments containing 109 A. excentricus cells/ml, 7.5, 8.2, and 4.5 third instar larvae were observed forCxquinquefasciatusAe. aegypti, and A. albimanus, respectively (Table I), and in the negative control (no food added) no mosquito larvae reached second instar in any of the mosquito species tested.

Transformation efficiency and expression of toxin genes by A. excentricus cells - Transformation of native A. excentricus C2 cells with plasmid pEA1 or pSOD2 was only obtained in cells treated with EMS. Transformation efficiency of A. excentricus C2 with plasmid pEA1 after treatment with EMS was 5.8x102 to 2.8x103 transformants/µg of DNA, 2 to 9 times lower than results obtained with A. excentricus strain 4724. However, transformation efficiency of A. excentricus cells with plasmid pSOD2 was 2.2x102 and 2.9x102 transformants/µg of DNA in A. excentricus strains 4724 and C2, respectively.

A. excentricus cells harboring plasmids pEA1, and pSOD2, and B. sphaericusBthuringiensis subsp. medellin, and recombinant B. thuringiensis grown as mentioned in materials and methods were boiled in Laemmli sample buffer and subjected to SDS-PAGE and Western blot. Proteins detected in the nitrocellulose membrane indicate that both A. excentricus clones produce proteins corresponding to the B. sphaericus binary toxin (Fig. 1, lanes 2, 4) as well as in B. sphaericus FWC culture (Fig. 1, lane 1). These two bands were absent in Asticcacaulis strains untransformed (Fig. 1, lanes 3 and 5). Expression of Cry11Bb toxin in A. excentricus recombinant strains was also observed (Fig. 2, lanes 3, 5). Although the band seen in these recombinant strains were not as strong as the one shown by recombinant B. thuringiensis strain (Fig. 2, lane 2), the molecular weight of the band corresponds to the original Cry11Bb toxin.

Bioassays - Toxicity (LC50) of recombinant A. excentricus strain C2 transformed with plasmid pEA1, expressing the B. sphaericus binary toxin was 1.0x105 and 1.08x106 cells/ml in Cx. quinquefasciatus and A. albimanus second instar larvae respectively (Table II). This strain was 11 and 7 times less toxic to Cx. quinquefasciatus and A. albimanus larvae, respectively, than final whole culture of wild type Bsphaericus. On the other hand, A. excentricus strain 4724 transformed with the same plasmid was 1.5 times less toxic to both mosquito larvae species than B. sphaericus. The A. excentricus recombinant strains (C2 and 4724) harboring plasmid pSOD2 did not show toxicity to first instar Cx.quinquefasciatus larvae in any of the concentrations tested (data not shown).

Since no toxicity was found in treatments containing A. excentricus cells transformed with plasmid pSOD2, we investigated whether A. ex-centricus protease extract could have degraded the Cry11Bb recombinant protein once it was exported to the cytoplasm. The Western blot shown in Fig. 3 indicates that the Cry11Bb protein is degraded to a fragment of ca. 39.5 kDa in all treatments including the Cry11Bb protein without A. excentricus strain 4724 protease extract (Fig. 3, lane 7). This same pattern was observed when different concentrations of Cry11Bb protein were incubated with cell extracts of A. excentricus strain C2 (Fig. 3, lanes 5 and 6).

Flotation experiments - Flotability is a desirable character in recombinant toxin-producing bacteria, since anopheline mosquito larvae feed in the top of the water column. A comparison of flotability of B. sphaericus and gram negative aquatic A. excentricus is shown in Fig. 4. Spores of B. sphaericus started to sediment after two days, and were completely sedimented six days later; however, cells of Aexcentricus transformed or untransformed remained in suspension, even six days after initiated the experiment.

 

DISCUSSION

An important point to be considered in developing recombinant bacteria for mosquito control is the ability of mosquito larvae to feed on a given bacterium host candidate. Pure cultures of A. excentricus strains C2 and 4724 were able to support mosquito larvae development in the three species tested. However, the degree of development reached in the mosquito species evaluated was significantly lower when compared to treatment with normal larval food (trout feed or liver powder), but higher than negative control, in which all larvae failed to reach second instar. Differences in larval growth between larvae in A. excentricus and normal larval food could be due to the kind of nutrients contained by each treatment. Merrit et al. (1992) have reported that mosquito larvae also ingest green algae, cyanobacteria, protozoa, and suspended organic material. Wotton et al. (1997) reported that development of A. albimanus larvae is directly related to the presence of suspended organic material as a diet complement. Differences in larval growth between the mosquito species could be due to specific nutrient requirements and/or to the specific ingestion rates for each mosquito species as indicated by Merritet al. (1992) and Thiery et al. (1993). The number of cells/ml of the A. excentricus cultures used to perform the feeding experiments resembles those found in mosquito larvae breeding ponds as reported by Grant and Long (1989). Once it was determined that A. excentricus strain C2 could support mosquito larvae growth, the transformation experiments were performed.

Transformation of native A. excentricus C2 cells with plasmid pEA1 or pSOD2 was only obtained in cells treated with EMS. Level of transformation efficiency of A. excentricus C2 with plasmid pEA1 after treatment with EMS is in correspondence to those reported by Tao et al. (1993), who only obtained transformants of Streptococcus mutans after treatment with EMS.

Expression of B. sphaericus binary toxin by recombinant strains of A. excentricus was confirmed by Western blot, as it was previously shown by Liu et al. (1996). Expression of Cry11Bb protein was also seen by Western blot, but the level of expression was low as compared to the expression of the B. sphaericus binary toxin.

The level of toxicity reached by native A. excentricus strains transformed with plasmid pEA1 is significantly greater than the toxicity obtained by unicelular cyanobacteria as reported by de Marsac et al. (1987), Xudong et al. (1993), and Sangthongpitag et al. (1997). In the same way, the LC50 of A. excentricus strain C2 transformed with plasmid pEA1 is two and four times higher than the toxicity obtained in previous reports by A. aquaticus and C. crescentus, respectively (Thanabalu et al. 1992, Yapet al. 1994b). A. excentricus recombinant strains (C2 and 4724) harboring plasmid pSOD2 did not show toxicity to first instar Cx. quinquefasciatus larvae in any of the concentrations tested (data not shown). Although the same vector was used to clone the B. sphaericus binary toxin gene and B. thuringiensis subsp medellinCry11Bb toxin gene, and both contain the identical promoter and transcriptional terminator sequences, negative results shown in the bioassays could be due to modifications caused by protease activity in the cytoplasm of recombinant A. excentricus strains, post-translational modifications, different codon usage of the host bacteria, or a combination of the above mentioned factors. We investigated if protease activity of A. excentricus cytoplasm extracts could be responsible for the lack of toxicity, and found that in the protease extract prepared by sonication of A. excentricus cells, as well as in the Cry11Bb control treatment, the 94 kDa protein was degraded to a 39.5 kDa fragment (Fig. 3), and this protein fragment was probably produced by activity of contaminant crystal proteases left during the sample preparation. Further experiments under way aimed to design new expression vectors for these gram negative aquatic bacteria will bring light on gene expression in these bacteria.

Results of flotability experiments of A. excentricus recombinant cells indicated that this characteristic could be possibly due to cell division that had occurred during the time of the experiment, and/or by movement mediated by flagella in those cells that reach the movile stage. Transformation of A. excentricus strains, and expression of the Bsphaericusbinary toxin by these recombinants did not affect the flotability properties of this bacterium.

 

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