The parasporal inclusion of Bacillus thuringiensis subsp. shandongiensis: Characterization and screening for insecticidal activity

JOURNAL

OF INVERTEBRATE

PATHOLOGY

59,

295-302

(19%)

The Parasporal Inclusion of Bacillus thuringiensis subsp. shandongiensis: Characterization and Screening for Insecticidal Activity PATRICIA V. PIETRANTONIO AND SARJEET S. GILL’ Department

of Entomology,

University

of California,

Riverside,

California

92521

Received June 24, 1991; September 25, 1991

INTRODUCTION The parasporal body of Bacillus thuringiensis subsp. shandongiensiswas characterized in terms of its structure, protein composition, and toxicological properties against several types of insects. The crystals of B. thuriugiensis shandougiensisappear to consist of a major protein of 144 kDa present in an spherical inclusion, as determined by transmission electron microscopy, tritration curve analysis, and SDS-PAGE of the solubilized crystals. A secondprotein of ca. 60 kDa is present in trace amounts and appearsto be associated with a small bar-shaped inclusion. The 144-kDa protein has been characterized by isoelectric point determination, N-terminal amino acid sequence analysis, amino acid analysis, and immunological crossreactivity. Its N-terminal amino acid sequence differed from that of other B. thuringiensis crystal proteins. The 144-kDa protein was not immunologically related to the crystal proteins of two toxic serovars (B. thuringieusis israelensis and B. thuringiensis kurstaki HD-1) and one nontoxic serovar (B. thuriugiensis indiana), as shown in immunoblots probed with antiserum raised against the 144-kDaB. thuriugieusis shandongiensis protein, the B. thuringiensis israelensis crystal proteins, and the trypsin resistant fragment of B. thuringiensis kurstaki Pl proteins. In contrast to most B. thuringiensis serovars, B. thuringieusis shandongiensiscrystals did not dissolve at pH 12. Solubilization was achieved in sodium bicarbonate at pH 8.3 and in the presenceof 25 mM dithiothreitol. The protein pattern of dissolved and undissolved B. thuringiensis shandongiensiscrystals after trypsin treatment did not differ from the controls, as observed by SDS-PAGE. Therefore, under the conditions of this study, the results suggestthat trypsin sites were not available on the 144-kDa protein. Crystals were nontoxic to larvae of the lepidopterans Trichoplusia ni, Spodoptera exigua, and Helicoverpa virescens or to the mosquitoes Aedes aegypti and Culex quinquefasciatus. 0 1992 Academic Press, Inc.

1 To whom correspondence dressed.

and reprint

requests should be ad-

Bacillus thuringiensis consists of a number of isolates, most of which are relatively specific, that are toxic only to a limited number of insect taxonomic groups: lepidopterans, dipterans, or coleopterans (Hofte and Whiteley, 1989). The toxicity of these bacterial isolates is due to their ability to produce proteinaceous parasporal inclusion bodies that upon ingestion are dissolved in the alkaline environment of the lepidopteran and dipteran larva midgut and are activated by proteolytic cleavage. Susceptible insects are killed by the destructive action of the endotoxin on the midgut epithelium, which produces leakage of the gut content, which causes disruption of the osmotic balance in the hemolymph, leading to paralysis and death (Liithy and Ebersold, 1981). Based on their biological activity B. thuringiensis strains can be divided into five pathotypes (Ellar et al., 1986). Four of the pathotypes are strains that are toxic to lepidopterans, dipterans, coleopterans, and dipterans and lepidopterans, respectively. The fifth pathotype is considered to be nontoxic. It is generally thought that B. thuringiensis isolates that produce parasporal bodies with no known insect toxicity are more extensively distributed than toxic ones (Ohba and Aizawa, 1986). Some of these contain proteins of 60 kDa in rhombic but flat crystals (Ohba et al., 1987). Noninsecticidal isolates of the serotype H 14 (B. thuringiensis israelensis) have been reported to contain crystal proteins with molecular masses of 2447 kDa. The lack of the 72- and 130-kDa proteins in these isolates in comparison with the type strain might be responsible for their lack of activity against dipterans. Further, it has been reported that although the crystals of the type strain and the nontoxic isolates are morphologically similar, their proteins are immunologically unrelated (Ohba et al., 1988). In another immunocharacterization study, antiserum raised against the 130-kDa Pl proteins of B. thuringiensis kurstaki

295 0022-2011/92

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296

PIETRANTONIO

cross-reacted with all the nontoxic crystal protein tested; however, the names of the strains were not clearly specified (Ellar et al., 1986). The need for information about B. thuringiensis crystal proteins with no known toxicity to insects may help in our understanding of the mechanisms of insect toxicity (Ohba et al., 1987). In addition, in reference to B. thuringiensis nontoxic subspecies, it is not known whether their crystals consist of mutant crystal proteins, unrelated proteins, or toxins that display a very narrow and as such, yet unknown host range (Whiteley and Schnepf, 1986). Among these apparently nontoxic isolates is B. thuringiensis shandongiensis serotype (H-22), which was isolated in China and contains a high molecular weight protein (Wang et al., 1986). At present, little information is available about the crystal proteins of B. thuringiensis shandongiensis and their insecticidal activity. The purpose of this study, therefore, was to provide some of the biochemical properties of its crystal proteins and to screen for biological activity against additional insect pests. In this report we show that the round-shaped parasporal inclusion of B. thuringiensis shandongiensis is composed of one major protein of ca. 144 kDa with acidic characteristics, whose NH,-terminal amino acid sequence differs from the reported sequences of all other B. thuringiensis parasporal proteins. An additional minor 60-kDa protein seems to be associated with a bar-shaped inclusion. The B. thuringiensis shuno!ongiensis 144-kDa protein does not appear to be related immunologically to the crystal proteins of two B. thuringiensis toxic strains (B. thuringiensis israeZensis and B. thuringiensis kurstuki HD 1) or one nontoxic strain (B. thuringiensis indiana). MATERIALS

AND

METHODS

Bacterial Strains. Lyophilized cultures of B. thuringiensis indiunu HD-521 and B. thuringiensis shandongiensis HD-1012 were obtained from the U.S.D.A. (Northern Regional Research Center, Preoria, IL). B. thuringiensis isruelensis was originally isolated from

the Institut Pasteur Standard 82 (IPS 82) and maintained in the laboratory. The isolate B. thuringiensis kurstuki HD-1, originally from the Forest Pest Management Institute (Sault Ste. Marie, Ontario, Canada), contains the CryIA a, b, c toxin genes reported for this strain (Moar et al., 1990). Culture conditions and isolation of crystals. Cultures of B. thuringiensis kurstaki, shandongiensis, and indiana were grown in sterile glucose-yeast-salts

(GYS) liquid media that contained 1 g glucose, 5 g yeast extract (Difco, Detroit, MI), 0.2 g MgSO,, 2 g (NH&SO,, 3 g K,HPOI, 50 mg MnSO,.HzO, and 60 mg CaCl, per liter. Cultures were incubated in an orbital shaker at 190 rpm and 28°C until complete lysis

AND GILL

was obtained, ca. 3 days. The cultures were harvested by centrifugation at 16,000g for 10 min at 4°C. Pellets of B. thuringiensis shandongiensis were washed with cold 0.5 M NaBr and distilled water, resuspended in distilled water, sonicated, and then separated by discontinuous sodium bromide gradients (25 to 56%, w/v). The gradients were centrifuged at 20,000 rpm in a SW 28 rotor for 1 hr at 4°C. B. thuringiensis kurstaki and indiana crystals were similarly separated by discontinuous sucrose gradients (67, 72, and 79%, w/v) and centrifuged at 24,000 rpm for 14 hr. Gradients were repeated three to four times until a good level of crystal purification was achieved as determined by light microscopy. For electron microscopy studies, crystals of B. thuringiensis shandongiensis were obtained from nonsonicated cultures grown on nutrient agar. Crystal solubilization. Solubilization of shandongiensis crystals was achieved by incubation in 50 mM

NaHCO,, pH 8.3, 25 mM dithiothreitol (DTT) at 37°C for l-3 hr. Using 1 mg of crystals for solubilization in 1 ml of buffer, 500 pg of soluble protein was obtained. B. thuringiensis kurstuki, indiana, and israelensis crystals were dissolved in 50 mM NaOH by a 30-min incubation at 37°C. Protein concentration was determined according to Lowry et al. (1951) using bovine serum albumin (BSA) as standard. For shandongiensis protein solubilized in the presence of D’IT the Bio-Rad (Richmond, CA) microassay was performed according to the manufacturer’s instructions. Gel electrophoresis, isoelectric electrofocusing, and tritration curve determination. SDS-PAGE was per-

formed using a discontinuous buffer system (Laemmli, 1970). The p1 was determined initially with a 3-9 pH gel and then with a 4-6.5 pH gel using the PhastSystern (Pharmacia, Uppsala, Sweden) according to manufacturer’s instructions. B. thuringiensis shandongiensis protein (180ng), solubilized in 50 mM NaHCOa, pH 8.3, and 6 mM DTT, was used per lane. Protein was loaded on the cathode or anode side of contiguous lanes. IsoGel p1 markers (FMC Bio Products, Rockland, ME) were used as standards. The tritration curve was performed in the PhastSystem on an IEF 3-9-pH-range gel. Production of polyclonal antibodies directed against the B. thuringiensis shandongiensis crystal protein. Solubilized B. thuringiensis shandongiensis crystal

protein was purified in 10% polyacrylamide-resolving gels by discontinuous SDS-PAGE. The ca. 130-kDa protein bands visualized by Coomassie blue were excised immediately, equilibrated to pH 6.8 with 0.125 M TrisiHCl, pH 6.8, 0.1% SDS, and 1 mM EDTA, and immediately subjected to a second electrophoretic separation as indicated above. The gels were then stained and destained and the protein bands excised from the gels were washed, mixed, and stored at -20°C until

THE

CRYSTAL

PROTEINS

OF B. thuringiensis

needed. The SDS-PAGE-purified protein was injected subcutaneously into New Zealand White female rabbits. The first two immunizations were administered in complete Freund’s adjuvant, while subsequent immunizations were given in incomplete Freund’s adjuvant. Immunizations were performed at 2-3 weekly intervals. A total of ca. 200-250 kg of protein was injected into each rabbit. Two weeks after the last injection blood was collected from the ear vein and the serum was separated. The level of sensitivity of this antiserum as determined by dot immunoblot was ca. 10 ng. Immunoblot. Protein bands separated by SDSPAGE were electrotransferred overnight onto nitrocellulose membranes (Heegaard and Bjerrum, 1988). After transfer, the membranes were washed for two 30min periods in phosphate-buffered saline containing 0.5% Tween 20 (PBST) and then with 1% BSA in PBST for 30 min. The membranes were then incubated with rabbit sera raised against shandongiensis-purified crystal protein, against kurstuki HD-159-kDa-purified tryptic resistant fragments from the 130- to 134-kDa Pl proteins, or against israelensis crystal proteins. The nitrocellulose membranes were then washed and developed as previously described (Blake et al., 1984). Trypsinization of B. thuringiensis and B. thuringiensis kurstaki HD-1

Crystals

and solubilized

ringiensis shandongiensis tuki HD-1 were subjected

shandongiensis crystal proteins. crystal proteins of B. thuand B. thuringiensis kurs-

to trypsin treatment by incubation at 37°C for 2 hr. Trypsin (EC 3.4.21.4.) Type XIII, from bovine pancreas and L-1-tosylamido-2phenyl(ethyl)chloromethylketone (TPCK) treated was dissolved in double distilled water (500 pg/ml). Crude soluble soybean trypsin inhibitor type II-S was prepared at the same concentration. To treat crystals with trypsin, the crystals were suspended in 50 mM NaHCO,, pH 8.3, and divided into loo-p1 aliquots containing ca. 100 kg (dry weight) of crystals. Ten microliters of trypsin solution was added to the crystals to give a trypsin to crystal protein (w:w) ratio of 1:20 for both strains. Solubilized crystal protein aliquots (100 ~1) were then subjected to trypsinization in a 1:5 (w:w) trypsin to soluble protein ratio. For shandongiensis 6 kg of soluble protein was used and 50 pg was used kurstaki HD-1. The soybean trypsin inhibitor was added in a 1:l ratio (inhibitor:trypsin (w:w)) after a 2-hr incubation period. Tubes were immediately cooled on ice and then analyzed by SDS-PAGE. Amino acid analysis and NH,-terminal amino acid sequencing of B. thuringiensis shandongiensis crystal protein. Samples were prepared in the same fashion for both analyses. B. thuringiensis shandongiensis-

solubilized protein was mixed with sample treatment buffer, heated at 60°C for 10 min, and subjected to elec-

297

shundongiensis

trophoresis in a 10% polyacrylamide gel using 0.1 mM sodium thioglycolate running buffer in the cathode reservoir (Hunkapiller et al., 1983). After electrophoresis, electrotransfer to 0.45 pm Immobilon (Millipore Co., Bedford, MA) was performed at 0.25 A for 3.5 hr. The membranes were then rinsed with water, stained with 0.1% Coomassie blue R-250 in 50% methanol for 5 min, destained in 50% methanol for 10 min, rinsed in water for 10 min, air dried, and stored at - 20°C. The protein bands were excised and subjected to pulse-liquid N-terminal protein sequencing (Applied Biosystems Model 475 A; Foster City, CA) and amino acid composition analysis with a microamino acid analyzer (Applied Biosystems 420-A-03) at the Biotechnology Instrumentation Facility at University of California, Riverside. Bioassays. Activity of B. giensis crystals was screened tobacco budworm Helicoverpa myworm Spodopteru exigua

thuringiensis

shandon-

on neonate larvae of the virescens or the beet ar(Dulmage et al., 1976), third instar larvae of the cabbage looper Trichoplusia ni (Dulmage et al., 19711, and fourth instar larvae of the mosquitoes Aedes aegypti and Culex quinquefasciatus (McLaughlin et al., 1984). Serial dilutions of a suspension of purified crystals were prepared based on the dried weight of the crystals. For larvae of S. exigua and H. virescens the mortality was recorded after 7 days (Dulmage et al., 1976), while for T. ni the mortality was recorded after 5 days (Dulmage et al., 1971). All bioassays were repeated on three different days. RESULTS

The crystals of B. thuringiensis shandongiensis were observed under phase-contrast microscopy to be irregularly shaped and spherical. The spherical crystals were readily isolated from the 44% (w/v) band of NaBr gradients and were ca. 1 p,m in diameter as observed by transmission and scanning electron microscopy (Fig. 1). The spherical crystal is enclosed within a multilaminated envelope similar to that observed in the nontoxic parasporal inclusion of B. thuringiensis neoZeonensis (Fig. la; Rodriguez-Padilla et al., 1990). Centrifugation of the sonicated crystal preparation in NaBr gradients showed the presence of a diffused white band at ca. 29% NaBr. This band appears to contain the bar-shaped crystal, which is on the outside of the spherical inclusion and its membrane, that separates from the spherical inclusion after pellet sonication. Preliminary results showed that the crystals were poorly soluble when boiled with SDS-PAGE sample treatment buffer containing 2-mercaptoethanol. The crystals were also insoluble in 50 mM NaOH, pH 12, with or without 25 mM DTT. Partial solubilization of the crystals was, however, achieved with 50 mM NaHCO,, pH 8.3, with 25 mM DTT. SDS-PAGE anal-

298

PIETBANTONIO

AND GILL

FIG. 1. Electron microscopy of purified B. thuringiensis shandongiensis envelope surrounds the spherical inclusion as indicated by a solid arrowhead. indicated by an open arrowhead. Magnification for both: 37,500 x

ysis of this solubilized fraction showed the presence of a protein with an estimated molecular mass of ca. 144 kDa in a 6% polyacrylamide gel. The mobility of this protein is less than that of the 130-kDa proteins of kurstaki HD-1 and indiana (Ellar et al., 1986) (Fig. 2A). SDS-PAGE analysis of the 29% NaBr fraction showed the presence of trace amounts of a BO-kDa protein that appears to be associated with the bar-shaped crystal (Fig. 1, Fig 2B). Figure 3A shows the SDS-PAGE of solubilized crystal proteins of kurstaki HD-1, shandongiensis, israeZensis, and indiana. In Fig. 3B, a nitrocellulose blot of these proteins was probed with the rabbit anti-144-

kDo

kDa

kDo

205-

/I 16 -97.4 -66

116-45 97.4

-

66-

-29 I

2

s

3

4

I

s

FIG. 2. SDS-PAGE Coomassie blue stained for the molecular mass determination of the B. thuringiensis shandongiensis crystal proteins. (A) 6% polyacrylamide gel. Lane 1: B. thuringiensis kurstaki HD-1 crystal proteins, 35 pg. Lanes 2 and 3: B. thuringiensis shandongiensis-solubilized crystal protein, 3 pg. Lane 4: B. thuringiensis indiana-solubilized crystal protein, 5 pg. (B) 10% polyacrylamide gel. Lane 1: B. thuringiensis shundongiensis crystals obtained from the 29% NaBr gradient band showing the presence of the 60kDa protein from the bar-shaped inclusion. S, molecular mass standards, 20 pg of protein.

crystals. (a) Transmission electromicrograph. A multilayer (b) Scanning electromicrograph. The bar-shaped inclusion is

kDa shandongiensis protein. The figure shows that there is little if any cross-reactivity with any of the proteins from the toxic strains, kurstaki HD-1 and isruelensis, or with the nontoxic proteins from indiana. As expected, the antibody shows a strong reaction to the 144-kDa shandongiensis protein (Fig. 3B, lane 3). The nitrocellulose membranes were also probed with antiserum against the B. thuringiensis israelensis crystal proteins and the 59-kDa toxic fragment from the kurstaki HD-1 proteins. These antisera did not cross-react with the 144-kDa protein from shandongiensis but reacted strongly with their respective antigens (Figs. 3C and 3D). The isoelectric point of the 144-kDa protein is ca. pH 4.4-4.6 (data not shown). The spherical crystals of B. thuringiensis shandongiensis apparently consist of only a single major protein because only one titration curve could be distinguished (Fig. 4). Analysis of the purified 144-kDa protein shows that relatively large amounts of aspartatejasparagine, valine, threonine, glycine, leucine, serine, isoleucine, and glutamate/glutamine are present (Table 1). Cysteine and cystine are not reliably analyzed by the methods used here. The N-terminal amino acid sequence of the 144-kDa protein is shown in Table 2. A comparison of the ability of shandongiensis crystals to undergo trypsinization shows that these crystals are fairly resistant to tryptic cleavage (Fig. 5, lane 4). In contrast, kurstaki crystals incubated with trypsin produced the expected trypsin resistant fragments of ca. 60-62 kDa (Fig. 5, lane 2). When the solubilized proteins were treated with trypsin, the 130- to 134-kDa proteins of B. thuringiensis kurstaki underwent rapid trypsinization (Fig. 5, lane 6). On the other hand, under an identical trypsin-to-protein ratio, the soluble B.

THE CRYSTAL PROTEINS

OF B. thurirzgiensis

kDa M

1205

/I 16 -97.4 -66 -45

kDa

‘we

299

shandunggiensis

kDa x ci

-I44

v

-I35 -65

,‘c*r; i

kDa

/

-130 i

k -29

-28

/ _“. jI; 2 3 4 Std. Std. 2 3 Std. I 3 I I23 4 Std. FIG. 3. SDS-PAGE and immunoblot analysis of B. thuringiensis kurstaki HD-1, B. thuringiensis israelensis, B. thuringiensis shundongiensis, and B. thuringiensis indiuna crystal proteins. (A) 12.5% polyacrylamide, Coomassie blue-stained SDS-PAGE. Lane 1: B. thuringiensis kurstuki HD-1, 50 p,g of protein. Lane 2: B. thuringiensis israelensis, 60 pg of protein. Lane 3: B. thuringiensis shandongiensis, 8 pg of protein. Lane 4: B. thuringiensis indianu, 4.5 pg of protein. Std, molecular mass standards, 20 pg. (B) Immunoblot with anti-144-kDa B. thuringiensis shandongiensis protein antiserum. A 12.5% polyacrylamide-SDS gel had been loaded with 1.7 pg of B. thuringiensis kurstaki HD-1 crystal proteins (lane l), 1.8 pg ofB. thuringiensis israelensis crystal proteins (lane 2), 1.4 pg ofB. thuringiensis shanobngiensis crystal proteins (lane 3), 0.8 pg of B. thuringiensis indiana crystal proteins (lane 41, and 20 pg of molecular mass standards (Std). (C) Immunoblot with anti-B. thuringiensis israelensis whole crystal antiserum. A 12.5% polyacrylamide-SDS gel had been loaded with 600 ng of B. thuringiensis israelensis crystal proteins (lane 2), 200 ng of B. thuringiensis shandongiensis crystal protein (lane 31, and 2 pg of molecular mass standards (Std). (D) Immunoblot with anti-59-kDa protein B. thuringiensis kurstuki HD-1 antiserum. A 10% polyacrylamide-SDS gel had been loaded with 467 ng of B. thuringiensis kurstuki HD-1 protein (lane I), 1 pg of B. thurirzgiensis shandongiensis protein (lane 3), and 1 p,g of molecular mass standards (Std). -

shandongiensis protein underwent slow proteolysis (Fig. 5, lane 8). The shandongiensis crystals were nontoxic to larvae of A. aegypti, H. virescens, and S. exigua, even at the highest dosages tested of 100 kg/ml for A. aegypti and a 500 pglg diet for the latter. Insignificant mortality was observed in T. ni larvae (6% at 500 kg protein/g diet) and C. quinquefasciatus (30% at 100 pg crystal protein/ml).

thuringiensis

ans, and toxic to dipterans, respectively. B. thuringienwere selected for comparison because they are spherical, composed of proteins of high molecular mass (ca. 130 kDa) (Ellar et al., 1986), and considered nontoxic (De Lucca et al., 1984; Whiteley and Schnepf, 19861, in a similar fashion to B. thuringiensis shandongiensis crystals (Wang et al., 1986). The B. thuringiensis shandongiensis crystals were not soluble at pH 12, the point at which effective solubilization of B. thuringiensis kurstaki, israelensis, and sis indiana crystals

DISCUSSION

B. thuringiensis shandongiensis crystals pared to those of B. thuringiensis indiana, giensis kurstaki HD-1, and B. thuringiensis which are nontoxic, toxic to lepidopterans

were comB. thurinisraelensis,

and dipter-

M9 t

Amino

B. thuringiensis

Amino acid ASX Glx Ser GUY His Arg Thr Ala Pl-0

FIG. 4. Tritration curve of the B. thuringiensis shandongiensis crystal protein. The IEF gel pH 3-9 was run in the first dimension to establish the pH gradient. For the second dimension run, 210 ng of protein obtained from crystals in the 44% NaBr gradient band was loaded along the middle line of the gel, between the anode ( + ) and the cathode ( - ) sides. The gel was run and silver stained following the manufacturer’s instructions.

Acid Composition

‘br Val Met Cys” Ile Leu Phe LYS Total

TABLE 1 of the 144-kDa Crystal subsp. shandongiensis %AA 11.2 7.3 7.7 8.3 1.4 3.3 8.6 7.0 6.5 5.9 8.8 0.7 0.2 7.3 8.2 4.4 3.1 100

Protein

of

Residues per mol 149 97 102 111 19 44 114 93 87 79 117 9 2 97 109 58 42 1329

a The methodology used does not allow for the precise determination of Cys.

300

PIETRANTONIO

Comparison of the N-terminal

GILL

TABLE2 Sequences of the B. thuringiensis subsp. shandongiensis144-kDa Crystal Protein with That of Published B. thuringiensis Crystal Proteins Position

1

2

3

4

5

Met

Asn

Met Met Met

l& Asp Asp Thr

Leu Asn Asn Asn Ser

Gln Asn Asn Asn Asn

Thr Pro Pro Pro Arg

GUY Asn Asn Asn LYS

Asn Val Val

Asn Leu Leu

Pro

Met

AND

6

from

the amino

7

end

Gene tw

8

9

10

11

12

13

GUY Ile Ile Be Asn

Val Asn Asn Asn Glu

Thr Glu Glu Glu Asn

Leu Cys CYS CYS Glu

His Ile Ile Ile Ile

Val Pro Pro Pro Ile

Glu Tyr Tyr Tyr Asn

CryIAk) CryIB

Gln Asn Asn

Asn Ser Ser

Gln GUY GUY

CYS Arg Arg

Ile Thr Thr

Pro Thr Thr

Tyr Ile Ile

Asn CYS CYS

C?yIC CryHA CryIIB

ASP

CyIA(a) CryIA(b)

Met

Glu

Met Met --

Am Asn

Glu Asn Ser

Met Met -Met --

Asn Asn Asn

Pro Ser

Asn Tyr GUY

Asn Gln Tyr

Arg Asn Pro

Ser LYS Leu

Glu Asn Ala

His Glu Asn

‘br Asp

Thr Glu Leu

Ile Thr Gln

LYS Leu GUY

CryIIIA CryIVA CryIVB

Met Met

Asn Glu

-Met

Glu

Pro Asp Asn

Tyr Ser Leu

Gln Ser Asn

Asn Leu His

LYS Asp CYS

Asn Thr Pro

Glu Leu Leu

‘b Ser Glu

Glu Ile Asp

Ile Val Ile

Phe Asn LYS

ClyIVC CryIVD CytA

indiana crystals was achieved. Optimal solubilization ofB. thuringiensis morrisoni “var. tenebrionis” crystals

occurs between pH 11 and 12.5 and crystals from most B. thuringiensis strains dissolve when the pH is raised above 9.5 (Bernhard, 1986). However, B. thuringiensis shundongiensis crystals dissolved at pH 8.3 in the pres-

ence of DTT. The low solubility

of B. thuringiensis nontoxic

crys-

kDa -I 16 X97.4 -66

I

2

3

4

5 6 7 8 std. B. thuringiensis shandongiensis

and B. crystals and dissolved crystal proteins (10% SDS-PAGE, Coomassie blue stained). Crystals’ trypsinization (lanes l-4). Lane 1: B. thuringiensis kwstuki HD-1 crystals’ control (100 pg) after 2 hr incubation at 37°C in 100 ~1 of 0.05 M NaHCO,. Lane 2: B. thuringiensis kurstaki HD-1 crystals (100 pg) after incubation in the presence of 5 p,g of trypsin. Lane 3: B. thuringiensis shandongiensis crystals control (100 kg) after 2 hr incubation. Lane 4: B. thuringiensis shandongiensis crystals (100 ug) after 2 hr incubation with 5 ug of trypsin. Other incubation conditions in lanes 2, 3, and 4 are as described for lane 1. Trypsinization of solubilized protein (lanes 5-8). Crystals were incubated in 0.05 M NaHCOa, 25 mM DTT for 3 hr. Insoluble material was removed by centrifugation and the supernatants were subjected to the following treatments. Lane 5: Solubilized B. thuringiensis kurstaki HD-1 crystal protein (6 kg) was incubated for 2 hr at 37°C in 0.05 M NaHCO,, 25 mM DTl’. Lane 6: Same as lane 5 but with the addition of ca. 1.2 pg of trypsin. Lane 7: Solubilized B. thuringiensis shandongiensis crystal protein (50 up) was incubated as described for lane 5. Lane 8: Same as lane 7 with the addition of 10 pg of trypsin. FIG.

5.

thuringiensis

Trypsinization

kurstaki HD-1

of

Ref. This work Schnepf et al. (1985) Wabiko et al. (1986) Adang et al. (1985) Brizzard and Whiteley (1988) Honee et al. (1988) Donovan et al. (1988b) Widner and Whiteley (1989) Sekar et al. (1987) Ward and Ellar (1987) Chungjatupornchai et aZ. (1988) Thorne et al. (1986) Donovan et al. (1988al Waalwijk et al. (1985)

tals at high pH has been previously reported. Crystals of two nontoxic isolates from Japan that contain a major protein of 60 kDa and several minor proteins are insoluble in 0.1 M bicarbonate buffer, pH 10.2, after incubation at 27°C for 2 hr (Ohba et al., 1987). These crystals solubilize upon the addition of silkworm gut juice but the solubility is much less than that of crystals of other B. thuringiensis strains (Ohba et aE., 1987). Although a 60-kDa protein is also present in B. thuringiensis shandongiensis, it is only a minor component of the parasporal inclusion. The titration curve of solubilized spherical crystals demonstrated that these crystals are composed of only one protein, as only one titration curve was observed. The isoelectric point (pH 4.4-4.6) revealed a crystal protein with acidic characteristics. The N-terminal sequence of this 144-kDa protein of shandongiensis has little in common with the published amino termini of B. thuringiensis crystal toxins. Only asparagine in the second position appears to be conserved since it is present also in CryIIA and B, CryIIIA and CryIVA, CryIVB and CryIVC proteins. The amino acid composition was compared to those of most reported B. thuringiensis crystal proteins. Among all, only the 27-kDa B. thuringiensis israelensis and the 144-kDa B. thuringiensis shandongiensis proteins possess a relatively high amount of valine, 10 and 8.8%, respectively. Furthermore, the amino acid composition of these two proteins is similar in the percentages of aspartate/asparagine, threonine, leucine, and isoleucine. However, immunoblots showed that the B. thuringiensis shandongiensis and B. thuringiensis israelensis antisera only reacted with their respective antigens. B. thuringiensis shandongiensis crystals have previously been shown to be nontoxic to larvae of Galleria

THE CRYSTAL

PROTEINS

OF B. thuringiensis

mellonella, H. armigera, Leucania separata, and C. pipiens (Wang et al., 1986). In this study no significant toxicity was observed against H. virescens, S. exigua, T. ni, Ae. aegypti, or C. quinquefasciatus. The insecticidal activity against T. ni was less than that reported for the mosquitocidal israelensis of 100 pg/ml of diet Ugnoffo et al., 1981). Insect toxicity and/or specificity of B. thuringiensis

crystal proteins depends among other factors on the activation of the crystal protoxins in the insect midgut (Haider et al., 1986; Jaquet et al., 1987). The gut juice of lepidopteran (Pritchett et al., 1981) and mosquito (Kunz, 1978) larvae is highly alkaline and contains proteases that exhibit optimal activity at high pH. Trypsin- and chymotrypsin-like activities have been demonstrated in the midgut of lepidopteran and mosquito larvae, with trypsin-like activity being predominant (Kunz, 1978; Nakamura et al., 1990). However, trypsin did not appear to significantly hydrolyze the 144-kDa protein nor yield any specific cleavage product. It is improbable that the experimental conditions used in this assay would have interfered with the complete digestion of B. thuringiensis shandongiensis protein. DTT did not appear to have inactivated the trypsin since the Pl proteins from the B. thuringiensis kurstaki control were completely digested, and the patterns were similar to those of the crystals incubated without DTT. The experimental pH 8.3 not only favored B. thuringiensis shundongiensis protein solubilization but also trypsin activity, which has an optimum pH range of 7-9. Similar conditions have been reported for the activation of B. thuringiensis berliner 1715 Pl protein (Convents et al., 1990). Furthermore, protoxins are degraded within the first 0.5 hr of incubation (Pfannenstiel et al., 1986; 1990). The activity of contaminant proteases in the B. thuringiensis shundongiensis crystals might have been responsible for the observed degradation. B. thuringiensis serine proteases and metalloproteases that hydrolyze the 8-endotoxins to large fragments (59-63 kDa) have been reported (Chestukhina et al., 1978; Kunitate, 1989). These are adsorbed to the crystals’ surface or enclosed in defects of the crystal lattice and show enhanced activity when the crystals dissolve (Chestukhina et al., 1978). Our observations may support the hypothesis that these proteins are not toxic because the crystals are neither easily solubilized nor trypsinized in the insect midgut. To date, only limited attempts have been made to investigate the relationship of nontoxic crystal proteins to the toxic ones. In this study, the 144-kDa B. thuringiensis shandongiensis protein antiserum did not cross-react with the crystal proteins of B. thuringiensis kurstaki HD-1, B. thuringiensis israelensis, or B. thuringiensis indiuna. In a similar fashion, the antisera against the B. thuringiensis israelensis crystals and B. thuringiensis kurstaki HD-1 proteins did not

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cross-react with the 144-kDa protein, indicating that these proteins are not related. In this work we presented evidence that the 144-kDa protein differs from the B. thuringiensis toxins reported so far. As the amino acid amino terminal sequence of the B. thuringiensis shandongiensis 144-kDa crystal protein had no similarity with those of previously reported B. thuringiensis toxins, this, together with its unusual biochemical properties and the lack of toxicity, suggests that this protein might be coded by a new B. thuringiensis crystal protein gene type. ACKNOWLEDGMENTS We thank B.A. Federici and J. Kitasako for transmission and scanning electron microscopy, respectively. The assistance of Y. M. Yu and S. M. Dai in providing purified crystals of B. thuringiensis isrueZensis and antiserum to B. thuringiensis israelensis crystals, respectively, and that of T. C. Baker, J. Trumble, G. Platner, and G. P. Georghiou of this department in providing insect cultures is gratefully acknowledged. Isolate HD-1 was kindly provided by William Moar of this department. This project was supported in part by BRSG 2 SO7 RR07010-24, awarded by the Biomedical Research Support Program, Division of Research Resources, National Institutes of Health. P.V.P. was supported by a F.F.H. scholarship from Rotary International Foundation. REFERENCES Adang, M. J., Staver, M. J., Rocheleau, T. A., Leighton, J., Barker, R. F., and Thompson, D. V. 1985. Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstuki HD-73 and their toxicity to Munducu se&cc. Gene 36, 289-300.

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