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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
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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
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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).
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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.
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