introduction and expression of the cry1ac gene of bacillus thuringiensis

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Introduction and Expression of the cry1Ac Gene ofBacillus thuringiensis

in a Cereal-Associated Bacterium, Bacillus polymyxa

S.N. Sudha, R. Jayakumar, Vaithilingam Sekar

Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India

Received: 12 August 1998 / Accepted: 25 September 1998

Abstract. The abilities ofBacillus polymyxa and Bacillus thuringiensis to survive on the rice phyllospere

were compared; it was found that B. polymyxa colonizes the crop better. This study also showed that B.

polymyxa inoculation to rice plants increased the shoot and the root growth of the crop. Efforts were made

to introduce the cry1Ac gene ofB. thuringiensis subsp. kurstaki into B. polymyxa so that the application of

such transgenic B. polymyxa strains would prove to be dually beneficial to rice crops both as a biopesticideand as a biofertilizer. Immunoblot analysis of the recombinant organism containing the cry1Ac gene, strain

BP113, indicated efficient expression of this gene in the heterologous host. Bioassays with the first instar

larvae of the yellow stem borer of rice (Scirpophaga incertulas) revealed that the protein preparations

from BP113 were toxic.

Bacillus polymyxa, a Gram-positive, endospore-forming,

diazotrophic organism found in association with several

grasses, is capable of atmospheric di-nitrogen reduction.

The effect of B. polymyxa inoculation on grasses like

Triticum, Lolium, Trifolium, and Agropyron has been

varied, ranging from increased shoot/root ratio [4],

increased seedling emergence [1], to higher yield and dryweight without significantly affecting plant development.

Chanway et al. [1] have attributed these effects to the

production of phytohormones like indole acetic acid by

the organism rather than to N2-fixation.

Bacillus thuringiensis (B.t.) is an important indus-

trial microbe owing to its ability to produce protein-

aceous, insecticidal, parasporal inclusions during sporula-

tion. The insecticidal crystal proteins (Cry proteins) are

solubilized in the larval midguts and are acted upon by

midgut proteases, which convert the Cry proteins into

activated toxins. These toxins bind to specific epithelial

receptors in the midgut and cause alteration of ion

channels and disruption of the membrane, finally result-

ing in larval death [3]. Several types of Cry proteins have

been isolated that are toxic to different orders of the insect

family. These crystal proteins, however, are found to be

highly unstable in the environment, and expression of the

highly AT-rich cry genes is very poor in unrelated plant

hosts [7]. To achieve efficient expression in plants,

extensive modifications (with respect to codon-bias) of

the cry genes have been found essential [2,8]. Since B.

polymyxa belongs to the same genus as B.t., problems

associated with the expression of cry genes in this host

would be minimal, and modifications of the gene would

be unnecessary.

Rice, an important cereal crop in the Asian subconti-

nent, suffers considerable damage from a serious lepidop-

teran pest, namely, the yellow stem borer (Scirpophaga

incertulas). Rice transgenic for the gene cry1Ab of B.t.

subsp. kurstaki (B.t.k.) was found to be tolerant to the leaf

roller Cnaphalocrocis medinalis and to the striped corn

borer Chilo suppressalis [2]. Developing transgenic crops

expressing the cry genes ofB.t. has been considered to be

a suitable approach to overcome the environmental

instability of the Cry protein. However, commercializa-

tion of transgenic crops is a time-consuming process.

Hence, we believe that application of Cry toxin on thesurface of the plants or expression of the Cry protein in

suitable phyllosphere-inhabiting organisms may be a

viable option for the prolonged delivery of the insecti-

cidal crystal proteins. B. polymyxa, which is well known

to show beneficial effects on wheat and other cereals

through colonization of their roots, has not been studied

in the context of paddy. Because our current studies

indicated that B. polymyxa colonized the phyllosphere ofCorrespondence to: V. Sekar

CURRENT MICROBIOLOGY Vol. 38 (1999), pp. 163167

An International Journal

Springer-Verlag New York Inc. 1999


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the rice crop better than B.t., we initiated efforts to

determine the persistence of B. polymyxa on rice phyllo-

sphere and the suitability of this organism as a delivery

system for Cry proteins.

Materials and Methods

Bacterial strains and growth conditions. B.t.k. strains HD73 andHD73-26 (Cry) were obtained from L.K. Nakamura (USDA, NRRL,

Peoria, IL, USA). B.t. subsp. sandiego and Escherichia coli strain

ECE53 were obtained from Bacillus Genetic Stock Center (Ohio State

University, Columbus, OH, USA). Strain ECE53 harbors a recombinant

plasmid pOS4201, which contains the cry1Ac gene of B.t.k. HD73

cloned as an end-filled NdeI fragment into the SmaI site of the vector

(Emr) pKK223-3. The broad-host range mobilizable cloning vector

pAT19 was obtained from Trieu-Cuot, France [12]. The B.t. strains were

grown in nutrient broth or L-Bertani (LB) broth with vigorous shaking

at 30C. Solid media were prepared by supplementing the respective

media with 1.5% agar. For sporulation, Spizizens minimal medium

(SCG) [1.52 g K2HPO4, 0.48 g KH2PO4, 0.2 g sodium citrate, 0.2 g

ammonium sulfate supplemented with 0.1% vitamin-free casamino

acids (acid-hydrolyzed casein), 0.5% glucose, and 1 mM MgSO4] was

used at 30C. B. polymyxa (ATCC842) was obtained from DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH (German

Collection of Microorganisms and Cell Cultures), Braunschweig,

Germany and was grown in glucose broth (GB, nutrient broth supple-

mented with 1% glucose). E. coli strains JM109 and JM110 (dam,

dcm) [13] and GM31 (dcm) were obtained from Escherichia coli

Genetic Stock Center, Yale University (New Haven, CT, USA). They

were maintained on LB agar plates or grown in LB broth at 37C.

Inoculation ofB. polymyxa and B. thuringiensis on rice seeds. A2-ml

overnight culture was used as inoculum for a 100-ml culture of the

organism. Cells grown to stationaryphase were harvested by centrifuga-

tion. They were washed once with sterile saline (0.85% NaCl) and

resuspended in sterile saline to give a final cell density of 107 CFU/ml.

The cell suspension (2 ml) was mixed thoroughly with surface-

sterilized rice seeds along with powdered activated charcoal and gum

arabic (1%) by gentle kneading in a polythene bag until all the seedswere uniformly coated. Inoculated seeds were placed on sterile petri

dishes containing moist filter paper discs (20 seeds/plate) and were

allowed to germinate. Root and shoot lengths were measured 5 days

after bacterial inoculation.

Plant inoculation and survival of the bacteria. Survival studies of the

bacteria were carried out on 4- to 6-week-old rice plants maintained in

pots. A culture (200 ml) of a spontaneous streptomycin-resistant(Str r)

mutant of B. polymyxa was grown in GB to stationary phase, and cells

were harvested by centrifugation. They were washed twice with sterile

distilled water and resuspended in sterile water to give a final

concentration of 107 CFU/ml. Gum arabic (1%) and Tween-20 (0.01%)

were added to the cell suspension. Cells were then sprayed on plants

(nearly 25 ml/pot). Zero-day counts were taken after the complete

drying of the sprays (i.e.,3 h). The survival of inoculated bacteria was

monitored at 5-day intervals for 3 weeks. About 20 seedlings from

different pots were picked randomly, and their leaf sheath and leaves

were cut into small pieces. The tissue-bound bacteria were freed by

immersing and gently agitating in 50 ml of sterile 100-mM MgSO4solution for 15 min. The washates were serially diluted and plated on

media containing appropriate antibiotics. The surviving population was

expressed as CFU/dry weight of plant material. Dry weights were

determined after drying the samples at 85C for 24 h. To determine the

persistence ofB.t. on rice, B.t.k. HD73-26 containing pBC16, which has

the tetracycline resistance gene as marker, was used. Cultures grown in

nutrient broth to stationary phase were used for spraying. Surviving

bacteria were monitored by plating washates of sprayed plants on

tetracycline-containing medium.

Bacterial cell suspensions were sprayed onto plants at 10-day

intervals, and the shoot and root lengths and dry weights of treated

plants were determined 6 weeks after the first inoculation. Control

plants were grown under the same soil and light conditions without

bacterial inoculation.

Transformation ofB. thuringiensis subsp. kurstaki and B. polymyxawith pATC11. The cry1Ac gene from the construct pOS4201 was

removed by digesting the plasmid with BamHI. In this construct, the

gene is flanked by two BamHI sites, one site 200 bp upstream of the

gene and another downstream in the multiple cloning site. Digested

DNA was separated on an agarose gel, and the 4-kb fragment containing

the cry1Ac gene was eluted from the gel by binding to DEAE-cellulose

membrane. This fragment was introduced into the unique BamHI site of

pAT19, and the recombinant plasmid was designated as pATC11. In

order to check the efficiency of expression of the cloned gene in a

Bacillus system, we selected the CryB.t.k. HD73-26 as a suitable host.

Competent cells of HD73-26 were subjected to electroporation in the

presence of 500 ng of pATC11 derived from E. coli strain JM110.

Electroporation was carried out with a Bio-Rad Gene Pulser at a field

strength of 1.2 kV/cm (resistance 200 ohms and capacitance 25 F).

One of the erythromycin-resistant (Emr) transformants was analyzed byslot-lysis gel electrophoresis [10] for the presence of pATC11.

Since attempts to introduce pATC11 by electroporation were

unsuccessful, the plasmid from E. coli was mobilized into B. polymyxa

with the assistance of a helper strain containing the broad-host-range

helper plasmid pRK2013. The Emr donor E. coli JM109 [pATC11], Kmr

helper E. coli MM249 [pRK2013], and Strr recipient (B. polymyxa)

were mixed,and cells from thetriparentalmating were diluted in 2 ml of

fresh LB and plated on medium containing erythromycin (8 g/ml) and

streptomycin (100 g/ml) to select for transconjugants.

Southern hybridization. Electrophoretic DNA transfer from agarose

gel to Zetaprobe membrane was done with Hoefer TE-70 Semiphor,

Semi-Dry transfer unit according to the manufacturers instructions.

DNA probe used for Southern hybridization was labeled with radioac-

tive [-32P]dCTP by random primer labeling with the oligolabeling kit

(Pharmacia LKB Biotechnology) according to the manufacturers

instructions.

Immunoblot of crystal proteins. Cells were grown on SCG minimal

plates until sporulation. The spore-crystal mixture was scooped out of

the plate and washed twice with 0.5 M NaCl followed by two washes

with distilled water. It was resuspended in distilled water containing 1

mM phenylmethyl sulfonyl fluoride and stored at 20C. Protein

concentration was determined according to Lowry et al. [6]. Sodium

dodecylsulfatepolyacrylamide gel electrophoresis (SDSPAGE) of

proteins was done according to Laemmli [5] with a Hoefer Mighty

Small vertical slab unit SE250.

Protein transfer onto nitrocellulose membrane was performed

with a TE70 Semiphor Semi-Dry transfer unit (Hoefer Scientific

Instruments, San Francisco, CA, USA) according to Towbin et al. [11].

The primary antibody was a polyclonal rabbit antiserum raised againstthe Cry1Ac protein of B.t.k. HD73. A goat anti-rabbit IgG antibody

conjugated with alkaline phosphatase was used as the secondary

antibody. The bound antigen-antibody-substrate complex was detected

with BCIP/NBT substrate.

Bioassays. Bioassays with S. incertulas were performed as follows.

Rice stem/sheaths from young plants were cut into pieces about 45 cm

long. The protein preparations of HD73 and BP113 were injected into

the stem with a syringe to give a final concentration of 50 ng/cm2 and

100 ng/cm2. This was keeping in mind the feeding habit of the young

larvae, which mainly feed on the young sheath/stem regions. Neonatal

164 CURRENT MICROBIOLOGY Vol. 38 (1999)


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larvae numbering six/replicate were allowed to feed on the treated

stem/sheath. All treatments were done in duplicates. Mortality of the

larvae was scored at 12-h intervals for up to 72 h.

Results

Effect of B. polymyxa inoculation on rice seeds and

plants. Inoculation of surface-sterilized rice seeds withB. polymyxa resulted in a 27% increase in root length of

5-day-old seedlings as compared with that of the un-

treated seedlings. Inoculation with B. thuringiensis also

showed an increase in root length of the seedlings (9%).

As a result of B.t. as well as B. polymyxa inoculations,

there was a marginal decrease (4% and 10% respectively)

in shoot length of the seedlings (not shown).

Inoculation of B. polymyxa on 1-month-old rice

seedlings resulted in an increase in root dry weight by

nearly 30% and shoot dry weight by 10% as compared

with uninoculated plants (Table 1). Inoculation with B.t.,

on the other hand, did not have any effect on the root and

shoot dry weights.

Survival of B. polymyxa and B. thuringiensis on rice

plants. The population of B.t. declined to about 5% of

initial counts within 5 days and decreased further to

0.2% after 10 days and to 0.08% on day 15 (Table 2). B.

polymyxa, on the other hand, showed a better survival

during the initial periods. The surviving population was

nearly 50% on the 5th day and thereafter came down to

about 1% by day 10 and to 0.6% on day 15.

Introduction and expression ofcry1Ac inB. thuringien-

sis HD73-26 and B. polymyxa. The authenticity of the

transformant HD73-26-113 containing pATC11 was con-firmed by restriction analysis and Southern hybridization

with radio-labeled 4-kb BamHI cry1Ac fragment of

pOS4201 (not shown).

Slot-lysis gel electrophoretic analysis of the selected

transconjugant of B. polymyxa, BP113, indicated the

presence of a large plasmid. Plasmid DNA prepared from

BP113 was subjected to restriction analysis and Southern

hybridization. These results confirmed the presence of

pATC11 in BP113 (Fig. 1).

Expression of the cry1Ac gene in B. thuringiensis

subsp. kurstaki and B. polymyxa. SDS-PAGE analysis

of proteins from sporulated cultures of both organismsrevealed the presence of the 132-kDa Cry1Ac polypep-

tide, which was absent in their respective hosts. The

132-kDa polypeptide produced by HD73-26-113 and

BP113 was confirmed as the Cry1Ac protein by immuno-

blot analysis. A few polypeptides of lower molecular

weight were also seen to cross-react with Cry1Ac antibod-

ies and were probably degradation products of the

132-kDa protein (Fig. 2 and 3). The extent of degradation

of the 132-kDa polypeptide in B. polymyxa appeared to

Table 1. Effect ofB. polymyxa inoculation on 1-month-old rice

seedlings

Uninoculated

(n 86)

Inoculated with

B. polymyxa (n 100)

Shoot dry weight (gm) 0.62 0.75 (15%)

Root dry weight (gm) 0.32 0.42 (30%)

Root/shoot ratio 0.51 0.56

Dry weights of rice plants that had been sprayed with B. polymyxa at

10-day intervals was determined for a period of 6 weeks. The shoots and

roots were weighed separately and the average weight was estimated.

Percentage difference in weight between control and treated plants is

shown in parentheses. n indicates the sample size.

Table 2. Survival ofB. thuringiensis and B. polymyxa on rice plants

Organism

Bacterial population on day after inoculation

(n 3) (CFU/g dry wt 103)

0 5 10 15

B. thuringiensis 186.00 8 9.20 1.6 0.42 0.1 0.16 0.03B. polymyxa 140.00 5 70.00 7 1.32 0.1 0.80 0.1

Fig. 1. Agarose gel electrophoresis of plasmid DNA (A) from BP113

digested with different enzymes. Plasmid DNA from BP113 was

digested with the following restriction enzymes: Lane 1, SalI; 2,

BamHI; 3, EcoRI. pATC11 (lane 4) was digested with SalI. The sizes of

the molecular weight markers are shown on the left margin. Southern

hybridization with the [-32P]-labeled BamHI fragment (4 kb) from

pOS4201 containing cry1Ac as a probe is shown in (B).

S.N. Sudha et al.: cry1Ac Gene of B. thuringiensis in B. polymyxa 165


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be minimal. The levels of Cry1Ac protein expression in

HD73-26-113 (Fig. 2) and B. polymyxa (Fig. 3) were,

however, lower than that in HD73.

Colonization of BP113 on rice. Six-week-old rice plants

previously sprayed with BP113 were used to study the

extent of colonization. The persisting bacteria from the

plant surfaces were enumerated by plating the leaf/stem

washates on medium containing streptomycin and eryth-

romycin. Surviving bacteria could be detected for up to 3

weeks, as seen with the untransformed B. polymyxa.

Bioassays. Feeding experiments with Scirpophaga incer-

tulas revealed that at the end of 36 h 50% mortality was

recorded at a concentration of 50 ng/cm2, and 66%

mortality at a concentration of 100 ng/cm2 with bothHD73 and BP113 treatments. All the larvae in the control

treatment were feeding normally and were found to be

active through the period of the bioassay. Mortality of

100% was recorded by the end of 72 h at a protein

concentration of 100 ng/cm2 with HD73 and BP113-

treated stems; at protein concentrations of 50 ng/cm2,

mortality of 83% and 75% was recorded with HD73 and

BP113-treated stems, respectively.

Discussion

An important criterion for choosing B. polymyxa as a

potential host for the delivery of cry genes is itsnitrogen-fixing ability and other beneficial effects it has

on its host grasses. In our study, inoculation on rice seeds

and plants showed overall increase in root and shoot

length (Table 1). The ability of B. polymyxa inoculum to

increase the root and shoot length of the wheat crop has

been attributed to indole acetic acid [1]. Although nif

genes have been identified in B. polymyxa, the increase in

biomass owing to N2-fixation seems unlikely, since the

phyllosphere would not be a suitable environment for

N2-fixation. The partly anaerobic conditions of the sheath

region, however, may provide an atmosphere conducive

for N2-fixation.

Studies on survival of B. polymyxa on rice indicate

the persistence of the organism for at least 15 days

post-inoculation. The ability of B. polymyxa to colonize

wheat rhizosphere has been attributed partly to the

production of antimicrobials such as polymyxins [9]. The

role of polymyxin in the ability of B. polymyxa to

colonize rice phyllosphere remains to be established.

In B.t.k., the cry1Ac gene directed the synthesis of a

132-kDa protein that cross-reacted with antibodies raised

against Cry1Ac from B.t.k. HD73. Degradation products

were visualized, with the 60-kDa toxic fragment being

the major one. In the transformant B. polymyxa BP113, a

similar expression of protein was also seen, althoughdegradation products were of lower Mr than seen in

HD73-26-113. SDSPAGE and immunoblot analyses

indicated that the 132-kDa polypeptide was more stable

in B. polymyxa than in B.t., since many low-molecular-

weight polypeptides that cross-reacted with the Cry1Ac

antibody were visualized in B.t.

The transgenic BP113 when sprayed on rice was

found to persist for up to 3 weeks at detectable levels.

Fig. 2. SDSPAGE analysis of crystal protein preparations of HD73-26-

113 (A). The sizes of the protein molecular weight standards in kDa are

given on the left margin (from top to bottom: myosin, b-galactosidase,

phophorylase b, fructose-6-phophate kinase, albumin, glutamic dehydro-

genase, ovalbumin, glyceraldehyde-3-phosphate dehydrogenase). Lane1, protein molecular weight standards; 2, HD73; 3, HD73-26; 4,

HD73-26-113. Immunoblot analysis of crystal protein of HD73-26-113

with Cry1Ac-specific antibodies is shown in (B). Lane 1, HD73; 2,

HD73-26; 3, HD73-26-113.

Fig. 3. SDS-PAGE analysis of protein preparations from sporulated

culture of BP113 (A). Molecular weight of the protein standards (lane 4)

in kDa is indicated on the right margin. Lane 1, untransformed B.

polymyxa; 2, BP113; 3, HD73; immunoblot analysis of the gel using

Cry1Ac-specific antibodies is shown in (B).

166 CURRENT MICROBIOLOGY Vol. 38 (1999)


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Enumeration studies of sprayed BP113 on rice indicated

that the level of persistence was about the same as that of

the wild-type strain and that the transgene did not affect

the survival of the organism on rice plants. The toxicity of

the crystal protein from BP113 was checked on neonatal

larvae of yellow stem borer and was found to be

comparable to that of B.t.k. HD73. The concentration ofcrystal protein required to achieve 100% mortality of

neonatal larvae ofS. incertulas in 72 h was 100ng/cm2.

In summary, the cloned cry1Ac gene in B. polymyxa

synthesized a polypeptide of 132 kDa that cross-reacted

with the Cry1Ac antibody. The transgenic BP113 was

toxic to larvae of S. incertulas. With its beneficial effects

on the rice crop and its better persistence than B.t. on the

rice phyllosphere, the use of BP113 as a biopesticide as

well as a biofertilizer seems promising.

ACKNOWLEDGMENTS

Financial support for this project was provided in part by the IndianCouncil for Agricultural Research, Govt. of India, through a grant-in-

aid (1-8/93-FCI). We thank Dr. M.G. Murty for helpful discussions

during the initial phases of this work. S.N. Sudha and R. Jayakumar are

grateful to the Council of Scientific and Industrial Research for a senior

and a junior research fellowship, respectively.

Literature Cited1. Chanway CP, Nelson LM, Holl FB (1988) Cultivar-specific growth

promotion of spring wheat (Triticum aestivum L.) by coexistent

Bacillus species. Can J Microbiol 34:925929

2. Fujimoto H, Itoh K, Yamamoto K, Kyozuka J, Shimamoto K (1993)

Insect resistant rice generated by introduction of a modified

-endotoxin geneofBacillus thuringiensis. Biotechnology 11:1151

1155

3. Hofte H, Whiteley HR (1989) Insecticidal crystal proteins of

Bacillus thuringiensis . Microbiol Rev 53:242255

4. Holl FB, Chanway CP, Turkington R, Radley RA (1988) Response

of crested wheatgrass (Agropyron cristatum L.), perennial rye grass

( Lolium perenne L.) and white clover (Trifolium repens) to

inoculation with Bacillus polymyxa. Soil Biol Biochem 20:1924

5. Laemmli UK (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227:680685

6. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein

measurement with folin phenol reagent. J Biol Chem 19:3265

3275

7. Murray EE, Rocheleau T, Eberle M, Stock C, Sekar V, Adang M

(1991) Analysis of unstable RNA transcripts of insecticidal crystal

proteins genes of Bacillus thuringiensis in transgenic plants and

electroporated protoplasts. Plant Mol Biol 16:10351050

8. Perlak FJ, Fuch RL, Dean DA, McPherson SL, Fischhoff DA

(1991) Modification of the coding sequences enhances plant

expression of insect control protein genes. Proc Natl Acad Sci USA

88:33243338

9. Rosado AS, Seldin L (1993) Production of potentially novel

anti-microbial substance by Bacillus polymyxa. World J Microbiol

Biotechnol 9:52152810. SekarV (1987) A rapid screening procedure for the identification of

recombinant bacterial clones. Biotechniques 5:1113

11. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of

proteins from polyacrylamide gels to nitrocellulose sheets: proce-

dure and some applications. Proc Natl Acad Sci USA 76:4350

4354

12. Trieu-Cuot P, Carlier C, Poyart-Salmeron C, Courvalin P (1991)

Shuttle vectors containing a multiple cloning site and a lac-Z gene

for conjugal transfer of DNA from Escherichia coli to gram-

positive bacteria. Gene 102:99104

13. Yannish-Perron C, Vierra J, Messing J (1985) Improved M13 phage

cloningvectors and hosts strains:nucleotide sequencesof M13mp18

and pUC19 vectors. Gene 33:103199

S.N. Sudha et al.: cry1Ac Gene of B. thuringiensis in B. polymyxa 167

introduction and expression of the cry1ac gene of bacillus thuringiensis

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