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INTRODUCTION |
Entomopathogenic nematodes of the genera Steinernema
and Heterorhabditis have been used for the biological
control of soil dwelling pests that include weevils and lepidopteran
species (1-3). Strong et al. (4, 5) have provided the best
illustration of entomopathogenic nematode predation in the environment
by their documentation of the ecological relationship between
Heterorhabditis hepialus and the ghost moth caterpillar,
Hepialus californicus. The mechanism by which the
entomopathogenic nematodes are able to predate and reproduce in the
host involves a mutualistic relationship between the nematode and its
symbiotic bacteria, Photorhabdus sp. and
Xenorhabdus sp. (6, 7). Bovien (8) had postulated an
association between a steinernematid species and a bacterium during the
1930's. However, it was not until 1966 that Poinar (9) reported that a
single species of bacterium in the family Enterobacteriaceae was
present in the anterior region of the infective nematode of this
species. Since then investigators have shown that
Steinernema species carry bacteria of the genus
Xenorhabdus while Heterorhabditis nematodes
harbor species of the genus Photorhabdus (10, 11). In
1993, Photorhabdus bacteria were proposed by Boemare and
colleagues (12) for re-classification as a distinct genus from
Xenorhabdus, based on a variety of phenotyptic, ecological, and molecular studies. Recently, increased support for the separation of these two genera was obtained employing 16 S ribosomal DNA analysis
(13). Photorhabdus is represented by a single species, Photorhabdus luminescens, so named because of its
bioluminescent nature. This phenotype is unique among the
Enterobacteriaceae and other bacteria of terrestrial origin.
Once an infective juvenile nematode has penetrated the host hemocoel,
the bacterial symbiont is released from the nematode gut, septicemia
becomes established, and insect death occurs within 48 h. Although
the nematodes may play a role in insect death, in most cases the
bacteria alone are sufficient to cause insect mortality following
injection into the hemocoel (14, 15). The importance of these bacteria
in the life cycle of the nematode has been well documented using
axenically reared nematodes (14, 16). Together with the lack of
evidence for the free living existence of this bacterium, it has been
postulated that the symbiotic association is essential for the survival
of both nematode and its symbiotic bacteria. However, one possible
exception is the report by Farmer et al. (17) who isolated
P. luminescens strains from clinical samples with no
apparent nematode association (18).
The precise set of mechanisms by which the symbiotic bacterium is able
to circumvent the defense host systems in the hemocoel is still under
investigation. It has been suggested that the virulence events that
lead to bacterial proliferation could involve multiple factors, such as
secretion of lipases and proteases, the release of lipopolysaccharide
molecules, and the anti-hemocytic properties of the bacterial cell
surface (19-28). Recently, Clarke and Dowds (29) speculated that a
secreted lipase is responsible for the insecticidal activity observed
against Galleria mellonella. In addition to
secreted factors, these bacteria are known to produce intracellular
inclusion bodies similar to those produced by Bacillus thuringiensis (30, 31). But unlike Bt endotoxin crystals they are
not insecticidal and are thought to provide amino acid nutrients for
the emerging nematodes (32). In other studies, Bowen (33) reported that
a soluble protein fraction derived from P. luminescens culture medium possessed sufficient insecticidal activity to kill Manduca sexta upon injection. As part of our efforts to find
suitable insecticidal proteins that could be employed to produce
insect-resistant plants, we initiated a study to further characterize
the nature of the oral insecticidal activity. It was found that of
P. luminescens W-14 fermentation broth showed excellent
potency against Southern corn rootworm
(SCR)1 neonates, a surrogate
maize pest. We describe here the isolation and characterization of two
distinct but structurally similar protein toxins that are highly potent
against SCR larvae. We also show that toxin could be further processed
and activated by protease cleavage.
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EXPERIMENTAL PROCEDURES |
Organism and Growth Conditions--
Stock inoculum of P. luminescens strain W-14 (ATCC accession number 55397) was produced
by inoculating 175 ml of 2% Proteose Peptone number 3 (PP3) (Difco)
liquid media with a primary variant subclone in a 500-ml flask and
incubated for 16 h at 28 °C on a rotary shaker at 150 rpm. The
production broth was achieved by inoculation of 1.75 ml of the stock
inoculum into fresh PP3 medium in 500-ml flasks (175 ml of
culture/flask). After inoculation, the culture was incubated at
28 °C for 24 h as above. Following incubation, the broth was
centrifuged at 2,600 × g for 1 h at 10 °C and
vacuum filtered through Whatman GF/D (2.7 µM) and GF/B (1 µM) glass filters to remove debris. The broth was then
used for the studies described here.
Southern Corn Rootworm Bioassay--
Protein fractions were
diluted into 10 mM sodium phosphate buffer, pH 7.0, and
applied directly in 40-µl aliquots to diet plate wells (surface area
1.5 cm2) containing artificial diet (34). The diet plate
was then allowed to air dry in a sterile flow-hood. The wells were then
infested with single, neonate Diabrotica undecimpunctate
howardi (Southern corn rootworm) hatched from surface sterilized
eggs. The plates were sealed, placed in a humidified growth chamber,
and maintained at 27 °C for 3-5 days. Mortality was then scored.
For quantitation of toxin potency, 16 insects per toxin dose were used,
and assays were repeated 2-4 separate times. LC50 was
determined using the toxin concentration needed to cause 50% insect mortality.
Purification of Insecticidal Activity--
Unless noted, the
isolation protocol entailed starting with 5 liters of broth that was
concentrated using an Amicon (Beverly, MA) spiral ultrafiltration
cartridge Type S1Y100 (molecular mass cut off 100 kDa) attached to an
Amicon M-12 filtration device. The retentate was diafiltered with 10 mM sodium phosphate, pH 7.0 (Buffer A), until the
absorbance of filtrate at 280 nm was below 0.1. The retentate was then
applied at 5 ml/min to a Poros 50 HQ (PerSeptive Biosystem, Framingham,
MA) strong anion exchange column (1.6 × 15 cm). The column was
washed with 5 bed volumes of Buffer A and then eluted with 0.4 M NaCl in Buffer A.
The biologically active fraction, determined by SCR bioassays, was
loaded in 20-ml aliquots onto a gel filtration column Sepharose CL-4B
(2.6 × 100 cm) which was equilibrated with Buffer A. The protein
was eluted in Buffer A at a flow rate of 0.75 ml/min. The biologically
active fractions were pooled and applied to a Mono-Q 10/10 column
equilibrated with 20 mM Tris-HCl, pH 7.0 (Buffer B), at a
flow rate of 1 ml/min. The column was washed with Buffer B until the
optical density at 280 nm returned to baseline level. The proteins
bound to the column were eluted with a linear gradient of 0 to 1 M NaCl in Buffer B at 2 ml/min for 60 min. Two-ml fractions were collected and activity was determined by a dilution series of each
fraction in the bioassay. Two activity peaks against SCR were observed
and were named A (fraction 14 to 19) and B (fraction 20 to 25).
Activity peaks A and B were pooled separately and both peaks were
further purified using the procedure described below.
Solid (NH4)2SO4 was added to the
above protein fractions containing either toxin A or B to a final
concentration of 1.7 M. Proteins were then applied to a
phenyl-Superose 10/10 column equilibrated with 1.7 M
(NH4)2SO4 in 50 mM
potassium phosphate buffer, pH 7 (Buffer C), at 1 ml/min. After washing
the column with 10 ml of Buffer C, proteins bound to the column were
eluted with a linear gradient of 1.7 M
(NH4)2SO4, 50 mM
potassium phosphate, pH 7.0, to 25% ethylene glycol, 25 mM
potassium phosphate, pH 7.0, at 1 ml/min for 120 min. Fractions were
dialyzed overnight against Buffer A.
The most active factions were pooled and applied to a Mono Q 5/5 column
which was equilibrated with Buffer B at 1 ml/min. The proteins bound to
the column were eluted at 1 ml/min by a linear gradient of 0 to 1 M NaCl in Buffer B.
For the final step of purification, the most biologically active
fractions were pooled, brought to a final concentration of 1.7 M with solid (NH4)2SO4,
and applied to a phenyl-Superose 5/5 column equilibrated with Buffer C
at 1 ml/min. Proteins bound to the column were then eluted with a
linear gradient of Buffer C to 10 mM potassium phosphate,
pH 7.0, at 0.5 ml/min for 60 min. Fractions were dialyzed overnight
against Buffer A.
The final purified protein from activity peak A and activity peak B
from the Mono Q 10/10 column was named toxin A and toxin B,
respectively. The purified toxins were found stable at 4 °C for over
6 months and were used for all the characterizations reported in this paper.
Determination of Native Molecular Mass--
The native molecular
mass was estimated by gel filtration on either a Superdex 200 HR 10/30
column or a Sepharose CL-4B column (1.6 × 50 cm) as specified in
each experiment. The columns were calibrated with a mixture of known
molecular mass standards.
Protein Determination, Gel Electrophoresis, and Western
Analysis--
Protein concentrations were determined according to the
method of Bradford (35) with bovine serum albumin as standard.
Native and SDS-PAGE analyses were performed on either 10% or 4-20%
gradient gels (36). Western blotting was performed using ECL Western blotting detection reagent according to the manufacturer's
instructions (Amersham).
NH2-terminal Amino Acid Sequencing of Purified Toxins
and Related Peptides--
The purified toxin A and toxin B, as well as
partially purified active gel filtration fractions, were separated on a
10% SDS-PAGE gel and blotted to a Bio-Rad polyvinylidene difluoride
membrane according to the manufacturer's procedure. The protein bands
were localized by staining with Amido Black for 1 min (0.1% Amido
Black 10 B (Sigma) in 10% acetic acid) followed by destaining for 1 min with 5% acetic acid. Blots were sent for NH2-terminal
sequencing at Cambridge ProChem (Lexington, MA).
NH2-terminal sequences are described under
"Results."
Determination of Molecular Weight by Matrix-assisted Laser
Desorption Ionization Time of Flight Mass Spectroscopy--
Protein
molecular mass using matrix-assisted laser desorption ionization time
of flight (MALDI-TOF) mass spectroscopy was determined on a Voyager
Biospectrometry workstation with delayed extraction technology
(PerSeptive Biosystems, Framingham, MA). Typically, the protein of
interest (100-500 pmol in 5 µl) was mixed with 1 µl of
acetonitrile and dialyzed for 0.5 to 1 h on a Millipore VS filter
having a pore size of 0.025 µM (Millipore Corp., Bedford,
MA). Dialysis was performed by floating the filter on water followed by
adding the protein/acetonitrile mixture as a droplet to the filter
surface. After dialysis, the dialyzed protein was removed using a
pipette and then mixed with a matrix consisting of sinapinic acid and
trifluoroacetic acid according to the manufacturers' instructions. The
protein and matrix (4 µl total volume) were allowed to co-crystallize
on a ~3 cm (2) gold-plated sample plate (PerSeptive Biosystems).
Excitation of the crystals and subsequent mass analysis was performed
using the following conditions: laser setting of 3050, pressure of
4.55e-07, low mass gate of 1500.0, negative ions off; accelerating
voltage of 25,000, grid voltage of 90.0%, guide wire voltage of
0.010%, linear mode, and a pulse delay time of 350 ns.
Production of Peptide-specific Antibodies--
The genes for
toxin A and toxin B described in this paper were subsequently cloned
(gene cloning to be presented elsewhere). Unique gene sequences were
selected for peptides A1, A2, and B2 (peptides to be described under
"Results"). The following peptides were synthesized according to
the deduced amino acids sequences: NPNNSSNKLMFYPVYQYSGNT (for peptide
A1), VSQGSGSAGSGNNNLAFGAG (for peptide A2), and FDSYSQLYEENINAGEQRA
(peptide B2). The corresponding antibodies to the above three peptides
were generated in Genemed Biotechnology Inc. (San Francisco, CA). The
crude sera were purified using a SulfoLinkTM Coupling Gel
column (Pierce). Each column was prepared by immobilizing a specific
peptide to the gel following the protocol described by the
manufacturer. The respective antisera was applied to the column at the
rate of 0.5 ml/min. The column was subsequently washed with
phosphate-buffered saline, pH 7.6. The purified antibodies were eluted
from the column using 0.05 M sodium acetate, pH 3.0, and
they were immediately neutralized to pH 7.0 with 1 M Tris, pH 8.0. The three purified peptide-specific antibodies were named synA1Ab, synA2Ab, and synB2Ab, and specifically recognized peptide A1,
A2, and B2, respectively.
Protease Treatment of Protoxin--
The standard reaction
consisted of 40 µg of purified P. luminescens W-14 toxin B
protease as specified under "Results," and 0.1 M Tris
buffer, pH 8.0, in a total volume of 100 µl. For control reactions,
protease was omitted. The reaction mixtures were incubated at 37 °C
overnight. At the end of the reaction, 10 µl was removed and then
boiled with an equal volume of 2 × SDS-PAGE sample buffer for
SDS-PAGE analysis. The remaining 90 µl of reaction mixture was
serially diluted with 10 mM sodium phosphate buffer, pH
7.0, and analyzed by SCR bioassay.
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RESULTS |
P. luminescens W-14 Insecticidal Activity Is
Proteinaceous--
The fermentation broth of P. luminescens
W-14 strain presented a broad spectrum of oral activity against a
variety of insects (data not shown), including SCR. Because of our
interest in controlling corn rootworm in maize, SCR bioassays
were used to follow the Photorhabdus insecticidal activity
in this study.
Consistent with prior studies by Ensign (33), it was found that the
majority of the activity was retained upon extensive dialysis or upon
concentration with devices containing 100-kDa molecular mass filters,
indicating that the majority of activity was associated with large
molecular mass material. Because of the large size, an Amicon M-12
filtration unit equipped with a 100-kDa membrane was used to enrich
activity from 5 to 10 liters of broth. Following a single step elution
from an ion-exchange column, the bulk of the activity was further
resolved by gel filtration using a preparative Sepharose CL-4B column
(Fig. 1A). The native molecular mass of the activity appeared to be in the range of 700 to
900 kDa, as judged by comparison to the migration of molecular weight
standards. SDS-PAGE analysis of this active protein fraction indicated
the presence of more than 10 major peptides (Fig. 1B).

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Fig. 1.
Separation and characterization of a large
molecular weight protein fraction from Photorhabdus W-14 broth.
A, separation of the active fraction by Sepharose CL-4B gel
filtration. The active fractions were pooled as indicated in the
figure. B, SDS-polyacrylamide 4-20% gel electrophoresis of
10 µg of the pooled active protein fraction (lane 2). The
molecular weight marker is indicated in lane 1.
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It was found that essentially all of the insecticidal activity could be
eliminated at temperatures above 60 °C or upon treatment with
protease K. Similar results were obtained with broth samples (data not
shown). These results are consistent with the toxic activity being
proteinaceous. In order to further determine the biochemical nature of
the protein toxin, samples were treated with a variety of reversible
and irreversible protease inhibitors which included E-64 (cysteine
inhibitor), 3,4-dichloroisocoumarin (serine inhibitor), leupeptin
(serine inhibitor), and pepstatin (aspartic inhibitor). In all cases,
no effects were observed on the biological activity of the toxin. In
addition, aliquots of toxin complex treated with inhibitors of
metalloenzymes, including 1,10-phenanthroline and EDTA, did not affect
the insecticidal potency.
As shown in Table I,
NH2-terminal amino acid analysis of selected peptides from
the SDS-PAGE (Fig. 1B) demonstrated that each was distinct,
but several peptides appeared to have substantial sequence similarity
(50-67%). The amino acid sequence for a 64-kDa peptide corresponded
to groEL, an Escherichia coli chaperonin (37),
whose concentration varied for each preparation. The occurrence of
related peptides suggested that several toxins may be present in the
860-kDa fraction.
Purification and Characterization of Two Individual Toxins from the
860-kDa Photorhabdus Fraction--
The 860-kDa fraction was applied to
a Mono Q column and resolved by a linear salt gradient. It was found
that the insect activity was broadly eluted throughout the gradient.
Each fraction was bioassayed by serial dilution in order to identify
those with the highest SCR activity. Two peaks were identified with
high SCR toxicity: one that eluted at approximately 0.2 M
NaCl (peak A) and the other at approximately 0.3 M NaCl
(peak B) (Fig. 2). Each activity peak was
pooled separately and further purified by a series of hydrophobic and
ion-exchange chromatography columns. The toxin purified from peak A was
denoted toxin A while the toxin from peak B was named toxin B. Both
purified toxin A and toxin B contained two predominant bands on a
4-20% SDS-PAGE (Fig. 3A). The peptides were named A1 and A2 for the large peptides, and A2 and B2
for the small peptides in toxin A and toxin B, respectively.

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Fig. 2.
Separation of two insecticidal protein
fractions by anion-exchange chromatography. Active fraction from
Sepharose CL-4B was applied to a Mono Q 10/10 column and eluted by a
linear NaCl gradient. Two active peaks against SCR, A and
B, were detected and pooled as indicated in the
graph.
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Fig. 3.
Characterization of isolated toxins.
A, 4-20% SDS-PAGE of purified toxin A (lane 1)
and toxin B (lane 2). 5 and 2 µg of protein were applied
to lanes 1 and 2, respectively. The protein
molecular weight marker is shown to the left of the sample
lanes. B, 2-15% native PAGE of purified toxin A
(lane 1) and toxin B (lane 2). 5 and 2 µg of
protein were applied to lanes 1 and 2,
respectively. C, estimation of the native molecular mass of
toxin A and toxin B by gel filtration on Superdex 200 10/30 column. The
protein markers used were: thyroglobulin, 670 kDa; bovine -globulin,
158 kDa; chicken ovalbumin, 44 kDa; equine myoglobin, 17 kDa; vitamin
B-12, 1.3 kDa.
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The subunit molecular weights of both toxins were assessed by MALDI-TOF
mass spectroscopy and SDS-PAGE as presented in Table II. When the SDS-PAGE gel was quantified
by densitometry, the relative band intensity of the large and small
peptides in both toxin preparations was found to be approximately 3 to
1 ratio, respectively. Given their molecular masses, these results
suggested that these subunits were represented in the purified toxins
in equal molar amounts. Toxin A and toxin B showed single stained bands
in native PAGE, with toxin A migrating further than toxin B (Fig.
3B). Using gel filtration on Superdex S-200, the native molecular mass of toxin A or toxin B was measured to be approximately 860 kDa (Fig. 3C). This was re-confirmed by gel filtration
on a Sepharose CL-4B column (data not shown). Treatment of the 860-kDa fraction with high salt or non-ionic detergents failed to affect the
mobility of the toxin activity on gel filtration. The insecticidal activity (LD50) of toxin A and toxin B against SCR was
determined to be 5 and 87 ng/cm2 diet, respectively (Fig.
4). Toxin A was also highly potent
against tobacco hornworm (M. sexta), a lepidopteran species,
however, for toxin B only growth inhibition was detectable against
tobacco hornworm.

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Fig. 4.
Estimation of biological activities
(LD50) of purified toxin A (solid circle)
and toxin B (open circle) against SCR by diet feeding
assays.
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Amino-terminal amino acid sequencing was performed on toxin A and
toxin B subunits. The NH2-terminal sequence of B1 was
identical to the sequence for the 201-kDa peptide presented in Table I, however, no amino-terminal sequence was obtained for the large subunit
of toxin A. High amino acid identity (~82%) was found between
the NH2 terminus of peptides A2 and B2, the smaller
peptides of the two toxins (Table
III).
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Table III
Comparison of NH2-terminal amino acid sequences of small
subunits of toxin A and toxin B
The symbols used are: |, identical amino acids; :, similar amino
acids.
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Since toxin B was an order of magnitude less active than toxin A
against SCR neonates, the possibility that the activity observed in
toxin B was due to a small amount of contamination of toxin A in the
final purified toxin B material was examined by Western analysis using
antibodies specific to synthetic peptides corresponding to either toxin
A or toxin B. Three peptide-specific antibodies used for analysis were
synA1Ab and synA2Ab, which specifically recognized peptides A1 and A2
in toxin A, respectively, and synB2Ab, which specifically recognized
peptide B2 in toxin B. Western analysis of purified toxin A and toxin B
(5 µg of protein/lane) was performed using the above three
antibodies. As shown in Fig. 5, immunological analysis showed that only
toxin A but not toxin B reacted when either synA1Ab or synA2Ab was used
(Fig. 5, A and B).
Conversely, only toxin B but not toxin A reacted when synB2Ab was used
(Fig. 5C). This indicated that no detectable
cross-contamination existed in the purified toxin A and toxin B
fractions. Therefore, the insecticidal activities observed for each
toxin were independent of each other.

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Fig. 5.
Western analysis of purified toxin A and
toxin B using peptide-specific antibodies. 5 µg of protein of
toxin A (lane 1) and toxin B (lane 2) was
applied. A, the blot was probed with synA1Ab as the primary
antibody. B, the blot was probed with synA2Ab as the primary
antibody. C, the blot was probed with synB2Ab as the primary
antibody.
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To eliminate the possibility that protease activity was responsible for
the insecticidal activity, samples of isolated toxins were examined for
enzymatic activity using fluorescein isothiocyanate-derivatized substrates. Compared with controls and endogenous proteases, no significant protease activity was found for either toxin fraction (data
not shown). A second biological activity that has been associated with
certain classes of bacterial toxins is the ability to lyse different
cell types. In order to examine the general lytic properties of these
toxins, in vitro assays were performed using rabbit
erythrocytes incubated with up to 0.7 and 2 µg of purified toxin A or
toxin B, respectively. Using protein sample buffer as controls, no cell lysis was found with either toxin, while erythrocytes treated with the
appropriate units of
-hemolysin from Staphylococcus aureus, a positive control, were completely lysed.
Processing and Activation of Toxin B--
In some cases, it was
noted that when Photorhabdus W-14 broth was rapidly purified
as described previously, a 281,040 kDa peptide (determined by MALDI-TOF
MS) was present in the final purified toxin B fraction as the major
component instead of the two peptides typically observed (Fig.
6A). It was also observed that
in some broth productions that toxin A was similarly present as a large
molecular weight species. The same purification protocols were used to
isolate the toxin B 281-kDa peptide from Photorhabdus W-14
broth as described earlier. Western analysis of the 281-kDa peptide
fraction using the peptide-specific antibody synB2Ab indicated that the
281-kDa peptide was related to the smaller toxin B peptide (Fig.
6B). The 58-kDa signal detected by the antibody indicated the presence of small toxin B subunit that was not readily visible by
Coomassie Blue staining. These data strongly suggested that the small
and large peptides were derived from a single 281-kDa peptide, a
protoxin peptide, possibly by protease(s) present in Photorhabdus W-14 broth. In diet bioassays, the 281-kDa
peptide was found to be approximately 10-fold less active than the
cleaved species.

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Fig. 6.
SDS-PAGE and Western analysis of toxin B
fraction as the 281-kDa peptide being the major component.
A, 4-20% SDS-PAGE of 5 µg of purified protein. The
molecular weight marker is indicated in the left lane. B,
Western analysis of 1 µg of purified fraction using synB2Ab as the
primary antibody.
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Two distinct metalloproteases, 38 and 58 kDa, have been purified from
W-14 fermentation broth (to be reported elsewhere). The effect of the
38-kDa metalloprotease on the 281-kDa peptides was evaluated in
vitro by incubation with the protoxin peptide (see "Experimental
Procedures"). In Fig. 7A,
the result of SDS-PAGE analyses of protease treatment of toxins are
shown. After overnight incubation, the 281-kDa material was converted
into 201- and 58-kDa peptides as judged by SDS-PAGE analysis. Western
analysis using synB2Ab verified that the 58-kDa peptide was generated
from the 281-kDa peptide (Fig. 7B). Bioassays of
protease-treated and untreated (control) toxin fractions showed that
the biological activity (LD50) against SCR was improved to
~150 ng/cm2 diet upon cleavage (Fig. 7C). The
58-kDa protease also had a similar effect on the cleavage and
activation of the 281-kDa peptide as the 38-kDa protease (data not
shown). These data demonstrated that the two peptides in toxin B
originated from a single protoxin of 281 kDa by protease cleavage.

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Fig. 7.
Cleavage and activation of toxin B fraction
as the 281-kDa peptide being the major component. A,
4-20% SDS-PAGE of untreated (lane 1) and 38-kDa
protease-treated (lane 2) toxin B fraction as the 281-kDa
peptide being the major component. 5 µg of protein was applied in
each lane. The molecular weight marker is indicated on the left.
B, Western analysis of untreated (lane 1) and 38-kDa
protease-treated (lane 2) toxin B fraction using synB2Ab as
the primary antibody. C, estimation of the effect of
cleavage by the 38-kDa protease on the biological activity
(LC50) against SCR. Solid circle, untreated
toxin B fraction; open circle, toxin B fraction treated by
38-kDa protease.
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DISCUSSION |
B. thuringiensis (Bt) endotoxins have been used as
sprayable microbial insecticides for nearly 3 decades with limited
success in the commercial marketplace (38, 39). The effective, reliable use of these proteins to control insect pests in the field remained elusive until advances in plant transformation technology allowed the
stable introduction of these genes in a variety of crop species. Today,
concerns about the development of insect resistance to Bt transgenic
plants and the narrow biological activity of Bt proteins have spurred a
renewed effort to discover orally active insecticidal proteins. Early
discovery programs focused on growth inhibitory proteins, such as
lectins and protease inhibitors (40-43). Their low oral activity and
lack of insect mortality proved to be a severe impediment to their use
in transgenic products, since very high levels of expression are
required for control. In general, the loss of insect control in
transgenic plants can be attributed to the premise that it is necessary
to maintain a pest control threshold which is sufficient to control at
least 90% of the insect pests during an active infestation. We have
arbitrarily defined the pest control threshold as the value of
expressed insect control protein (E), in units of micrograms
per milligram of soluble protein, divided by toxin potency
(P), whose units are micrograms required for LD90 in
bioassays; pest control threshold = E/P. Therefore, it
is advantageous to discover oral protein toxins that are highly efficacious, resulting in high pest control threshold values. Such
toxins may prevent failures due to reduced expression as a result of
environmental factors such as stress, senescence, or other
physiological events. However, the idealized pest control threshold
value required for plant resistance is currently being defined as
scientists evaluate efficacy of Bt and other pest control transgenic
plants in the field.
Although the availability of orally active insecticidal proteins
outside the Bt endotoxin family that meet the efficacy hurdles required
for pest control has been limited to date, several academic and
industrial laboratories have reported interesting new leads. For
example, agricultural companies such as Novartis have discovered several new bacterial toxins referred to as VIPs, or vegetative insecticidal proteins, from a variety of Bacillus species.
One, VIP3A, an 88-kDa protein, has excellent activity against black cutworms and armyworms, both lepidopteran pests (44, 45). Another
bacterial toxin includes cholesterol oxidase derived from Streptomyces broth, that has shown to have selective, high
potency against cotton boll weevil (46). The engineered cholesterol oxidase gene has been transformed into cotton plants and is currently under evaluation for insect resistance. Bowen (33) first reported the
presence of a novel, very large protein derived from the bacterial genus, Photorhabdus, that was orally toxic to M. sexta. It is too early to know whether the second wave of
insecticidal proteins will be able to withstand insect pressure in the
field and provide the desired insect control to be commercially
competitive with chemical sprayable products.
We have demonstrated here that a high molecular weight protein fraction
can be routinely isolated from the fermentation broth of P. luminescens strain W-14. This protein fraction represents the
majority of insecticidal activity in the fermentation broth and
contains at least 10 distinct peptides that can vary with each
fermentation. The 860-kDa fraction was shown to be sensitive to heat
and protease K treatment, suggesting that it is proteinaceous in nature
and distinct from other reported insecticidal agents such as
lipopolysaccharides, which are stable at higher temperatures (47).
Amino acid sequencing of the NH2 terminus of partially purified toxin peptide fractions indicated that each peptide is distinct; however, several peptides have significant homologies. These
results suggest that there may be several genetically related toxins
produced by the P. luminescens strain W-14, and therefore, the partially purified fraction (Fig. 1) represents a complex of
several toxins. Evidence for this hypothesis was supported by the
discovery of two remarkably similar but distinct SCR protein toxins
that are composed of equal molar amounts of a small and large peptides.
Amino-terminal analyses of the small subunits suggested that the toxins
are highly related in their amino acid identity (82%) and similarity
(91%). In Table I, a third peptide, 61 kDa had 50% homology over a 10 amino acid sequence with both small peptides of toxin A and toxin B. This suggests the possibility that other toxin(s) are present in the
toxin complex. This finding together with the broad elution profile of
insect activity from ion exchange resin has led to continued
investigation of other related protein toxins. The presence of multiple
toxins is not surprising, based on observations that B. thuringiensis organisms typically contain more than one endotoxin
(48, 49). The production of multiple toxins by Photorhabdus
organisms may be a necessary attribute to assure survival of the
juvenile nematodes whose host is vulnerable to predation by other
insects and nematodes.
The isolated protein toxins were unusual in their gross protein
biochemical properties with respect to other insecticidal proteins
described previously. Most notably, unlike Bt endotoxins which are monomeric with molecular sizes ranging from 25 to 130 kDa,
both Photorhabdus toxins described appear to be very large oligomers, perhaps tetramers, of approximately 860 kDa. Furthermore, the protein toxin is highly soluble at neutral pH environments, in
contrast to Bt, where the toxin molecules natively exist in crystalline
inclusion bodies (48). Other biochemical aspects, such as the large
sizes of toxin subunits, ~280 kDa for the protoxin, are not unusual.
For example, protein subunits for Clostridium difficile
toxins A and B have been calculated to have molecular masses of 308,057 and 269,709 Da, respectively (50). Furthermore, Photorhabdus
toxins appear to have similarities to other generic A-B type bacterial
toxins that are proteolytically processed to the mature form (51).
Interestingly, the Photorhabdus broth contains two distinct
metalloproteases that are capable of specifically cleaving the native
protoxin into large (208 kDa) and small (63 kDa) molecular mass
subunits that are not held together via a disulfide bridge (data not
shown). The two subunits added together, ~271 kDa, did not account
for the total molecular size of the protoxin, 281 kDa. Also, a 216-kDa
species was observed in protease-treated samples, suggesting that
additional trimming of the larger subunit occurs (data not shown). Both
the unprocessed and processed species had a similar native molecular
size, but the potency of the oligomeric protoxin B was 5-10-fold less
active than fermented or in vitro processed species. These
results suggest that the toxin molecule is first assembled as an
oligomer in the bacterial cell or during secretion, and subsequently
processed to the mature form in the media. It is of interest to note
that one peptide that has been observed in the 860-kDa toxin fraction
is a homolog of GroEL, a chaperonin (37). However, this
chaperonin homolog was not present in the purified toxins, and
therefore, not necessary for toxicity.
The potential similarity to other bacterial toxins that are known to
contain enzymatic subunits provided the impetus to perform a set of
experiments to examine the toxins for proteolytic activity. For
example, the hemolysin toxin from Erwinia chrysanthemi is known to contain a metalloprotease moiety (52). However, no proteolytic
activity of the Photorhabdus toxins, either the partially purified fraction or purified toxins, was observed using a variety of
protease substrates. Furthermore, the use of irreversible and reversible inhibitors to metal ions and serine and cysteine residues failed to inactivate to the insect activity. These results indicate that the toxin does not appear to be a protease or an enzyme with a
serine or cysteine at the active site. Furthermore, we examined the
toxin for hemolytic activity which is known for certain types of
bacterial toxins and found that neither toxin A or B adversely affected
rabbit erythrocytes in vitro (data not shown). In
toto, these studies indicate that some general biochemical
features appear to be related to bacterial toxins, but at this time no definitive correlation was observed. The genetic relationship of these
toxins to other bacterial toxin genes will be addressed in a separate
article describing the isolation of their genes.
We further examined the effects of other protease preparations, such as
trypsin and a variety of crude protein extracts from guts of several
species of insects, on the 281-kDa peptide. Under appropriate
conditions, the 281-kDa peptide can be processed into two peptides in
the similar fashion as observed with the purified metalloproteases as
judged by SDS-PAGE. However, it was also observed that treatment with
different insect gut protease preparations had differential effects on
the insecticidal activity against SCR, ranging from loss of activity to
more than 10-fold activation. This suggests that the precise cleavage
of the protoxin by proteases impacts the degree of insecticidal
activity. Furthermore, we have observed similar effects on toxin
activation upon treatment with plant-derived protease preparations.
Currently, we are using mass spectrometry to dissect the molecular
differences among cleavages by different proteases to elucidate the
precise mechanism of activation. In conclusion, to date these data
suggests that there is a general proteolytically sensitive region is
present in the protoxin that is accessible to a variety of proteases,
however, it is unclear whether different proteases affect the degree of
toxin activation upon cleavage.
One aspect of these protein toxins that is unique is their high potency
against corn rootworm species, including Western corn rootworm (to be
reported elsewhere). Both toxin A and toxin B had high potency against
SCR, 5 and 87 ng/cm2 diet, respectively, which is in the
range of the most potent Bt toxins against lepidopteran species. In
contrast, Bt endotoxins have been reported to have relatively low
activity against corn rootworm species, LC50 of 10 to 1000 µg/cm2 diet (53). In addition, toxin A was also found to
have good efficacy against tobacco hornworm, a lepidopteran insect.
This activity range is unusual, given the general inability of Bt
toxicity to cross insect orders (48). We have also determined that
insecticidal activity of W-14 Photorhabdus toxins extends to
several other insect orders beside coleopteran and lepidopteran (data
not shown).
The high potency of both toxins make them attractive candidates as
potential insect control genes for transgenic plants. Another advantage
for transgenic applications is the finding that a single protoxin was
responsible for the insect toxicity, suggesting that only a single gene
may be required for biological activity. Our gene cloning results and
heterologous expression studies (to be reported elsewhere) corroborated
the biochemical studies described herein. Recently, Bowen et
al. (54) have described the isolation and cloning of similar toxin
proteins and genes by alternate methods. Their results are also
consistent with the observations reported in this article. Furthermore,
gene disruptions of proteins identical to toxin A and toxin B directly
reflected the biological potencies of the two toxins against tobacco
hornworm (54). In the process of our collaborations, we have agreed to
use a standardized nomenclature for the toxins discovered. Toxin A and
toxin B described here are identical, based on our cloned genes, to
peptides denoted TcdA and TcbA (54), respectively. Cumulatively, these
research data verify that the purified toxins reported here are
critical components of insecticidal toxic activity observed in
Photorhabdus broth.