Insecticidal Toxins from Bacillus thuringiensis subsp. kenyae - NCBI

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1991, P. 0099-2240/91/020349-10$02.00/0 Copyright © 1991, American Society for Microbiology

349-358

Vol. 57, No. 2

Insecticidal Toxins from Bacillus thuringiensis subsp. kenyae: Gene Cloning and Characterization and Comparison with B. thuringiensis subsp. kurstaki CryIA(c) Toxins MICHAEL A. VON TERSCH,* HELEN LOIDL ROBBINS,t CHRISTINE S. JANY, AND TIMOTHY B. JOHNSON Ecogen Inc., 2005 Cabot Boulevard West, Langhorne, Pennsylvania 19047 Received 24 July 1990/Accepted 15 November 1990

Genes encoding insecticidal crystal proteins were cloned from three strains of Bacillus thuringiensis subsp. kenyae and two strains of B. thuringiensis subsp. kurstaki. Characterization of the B. thuringiensis subsp. kenyae toxin genes showed that they are most closely related to cryIA(c) from B. thuringiensis subsp. kurstaki. The cloned genes were introduced into Bacillus host strains, and the spectra of insecticidal activities of each Cry protein were determined for six pest lepidopteran insects. CryIA(c) proteins from B. thuringiensis subsp. kenyae are as active as CryIA(c) proteins from B. thuringiensis subsp. kurstaki against Trichoplusia ni, Lymantria dispar, Heliothis zea, and H. virescens but are significantly less active against Plutella xylostella and, in some cases, Ostrinia nubilalis. The sequence of a crylA(c) gene from B. thuringiensis subsp. kenyae was determined (GenBank M35524) and compared with that of crylA(c) from B. thuringiensis subsp. kurstaki. The two genes are more than 99% identical and show seven amino acid differences among the predicted sequences of 1,177 amino acids.

During sporulation, many strains of Bacillus thuringiensis produce protein inclusions that are lethal to lepidopteran, dipteran, or coleopteran insects. Lepidoptera-active toxins are generally produced as protoxins that are solubilized in the alkaline midgut of susceptible larvae. The solubilized protoxin is proteolytically activated to the mature toxin by proteases present in the larval midgut or naturally associated with the protoxin. Many of the cry genes encoding these insect toxins have been cloned and characterized (for a recent review, see reference 19). Cry proteins are classified by structural relatedness and insect toxicity spectra (19). Among the Lepidoptera-active cryI class, six different genes, the products of which share at least 50% amino acid identity, have been recognized. Three of these six were initially categorized as the 4.5-, 5.3-, and 6.6-kb genes on the basis of the size in kilobase pairs of a HindlIl fragment containing the 5' end of the gene (24). These three genes, cryIA(a), cryIA(b), and cryIA(c), respectively, are greater than 80% identical in deduced amino acid sequence and are therefore considered subclasses of cryIA (19). The divergences of these sequences are concentrated in the central core of the proteins and are largely retained in the proteolytically activated toxins (17). Despite the substantial relatedness of the crylA genes, different representative gene products of each of the three subclasses exhibit markedly different levels of insecticidal activity against susceptible lepidopteran insects (12, 19). In this report, we describe the identification, cloning, and characterization of insecticidal toxin genes from three different strains of B. thuringiensis subsp. kenyae; these genes correspond to novel hybridizing HindIll fragments. We report the DNA sequence for one of these genes and demonstrate that this gene is highly related to cryIA(c) from

B. thuringiensis subsp. kurstaki strains. We also report that the insect toxicities of the CryIA(c) proteins of each subspecies are equal for some lepidopteran insects but significantly different for others. MATERIALS AND METHODS

Bacterial strains. The bacterial strains used in this study described in Table 1. Plasmid complements in wild-type and derivative B. thuringiensis strains were visualized after electrophoresis of plasmids from cells lysed in the wells of agarose gels (modified Eckhardt lysates [13]). Plasmid masses were determined by comparison with plasmid mobilities in B. thuringiensis subsp. kurstaki HD1. Materials. Media for routine cultivation of bacteria were obtained from Difco Laboratories, Detroit, Mich. LB (28) broth and plates were used for the cultivation of both Escherichia coli and Bacillus strains. Antibiotics and other chemicals were from Sigma Chemical Co., St. Louis, Mo. E. coli transformants were selected and maintained on media containing 50 ,ug of ampicillin per ml. Bacillus transformants were selected and maintained on media containing either 10 ,ug of tetracycline per ml or 5 ,ug of chloramphenicol per ml. Renografin-76 was obtained from Squibb Diagnostics, New Brunswick, N.J. Restriction and DNA-modifying enzymes were from Life Technologies Inc., Gaithersburg, Md., New England BioLabs, Beverly, Mass., or Promega Corp., Madison, Wis., and were used in accordance with the manufacturer's recommendations. A DNA sequencing kit (Sequenase) was obtained from United States Biochemical Corp., Cleveland, Ohio. Preparation of DNA. Vector DNA was purified from E. coli HB101 by an alkaline lysis technique (28) modified so that solution I did not contain lysozyme. Bacillus vector plasmids that do not replicate in E. coli were purified from B. megaterium VT1660 by the following modification of a Sarkosyl lysis technique (5). B. megaterium strains were grown overnight at 30°C and 200 rpm in 500 ml of LB broth are

* Corresponding author. t Present address: Merck Sharpe & Dohme Research Laboratories, West Point, PA 19486.

349

350

APPL. ENVIRON. MICROBIOL.

VON TERSCH ET AL. TABLE 1. Bacterial strains Construction

Source

Strain

Plasmid masses (MDa)

B. thuringiensis subsp.: kenyae HD63 kenyae HD588 kenyae HD617 kenyae HD588-2 kurstaki HD73-26 kurstaki HD73-26-40 kurstaki HD73-26-45 kurstaki HD73-26-10 kurstaki HD263-6 kurstaki EG2069

5.2, 5.3, 8.5, 12, 40, 49, 94, 125, -140 4.9, 5.3, 65, 125, -140 4.9, 5.2, 61, 115, -140 4.9, 5.2, 125, -140 4.9 4.9, 61 4.9, 5.3, 65 4.9, 44 Not determined Not determined

B. megaterium VT1660

None

38

E. coli HB101

None

4

supplemented with the appropriate antibiotics. The cells were harvested by centrifugation and resuspended in a one-fifth volume of 50 mM Tris (pH 8)-10 mM EDTA containing 0.1 mg of RNase per ml and 1.0 mg of lysozyme per ml. The cells were incubated at 37°C for 60 min, Sarkosyl was added to a final concentration of 0.1%, and the cells were lysed by gentle inversion of the cultures. The lysates were centrifuged, and the supernatants were extracted with phenol and chloroform. The plasmids were precipitated from the aqueous phase with ethanol, and the pellet was resuspended in 3.0 ml of TE buffer (28). Vector plasmids were further purified by equilibrium centrifugation in CsCl and ethidium bromide gradients. Genomic DNA for cloning experiments was isolated from B. thuringiensis strains by spooling the DNA from the interface generated after layering ethanol above the Sarkosyl lysates. Plasmid miniprep DNA from E. coli transformants was isolated by the boiling method of Holmes and Quigley (20). Cloning of cryIA genes. In general, a Southern blot (28) of a series of restriction digests of B. thuringiensis genomic DNA was probed with an N-terminal intragenic 726-bp EcoRI fragment of cryIA(a) isolated from pES1 (34) to identify single hybridizing fragments large enough likely to contain entire cryIA genes. Restriction fragments of the appropriate sizes were subsequently purified from preparative agarose gels by electroelution and ligated to either E. coli vector pBR322 or E. coli and Bacillus vector pEG7 (39). The ligation products were used to transform competent cells of E. coli HB101. Positive clones were identified among E. coli transformants by colony hybridization (14) to the cryIA(a) probe and by mortality in qualitative bipassays of neonate larvae of Heliothis virescens. Quantitative bioassays of CryIA(c) proteirw against lepidopteran insects. B. megaterium strains harboring individual crylA(c) genes were grown for 3 days at 30°C on NSM plates (26) containing either tetracycline or chloramphenicol as appropriate. Cells were recovered from plates, pelleted, and resuspended in TNT buffer (50 mM Tris hydrochloride [pH 7.5], 100 mM NaCl, 0.05% Triton X-100) containing 1 mg of lysozyme per ml. Preparations were incubated at 37°C for 1 h and briefly sonicated to ensure complete lysis. Spore and crystal mixtures were washed once with TNT buffer to remove lysozyme and resuspended in TNT buffer. Crystal inclusions were separated from spores and cell debris by equilibrium centrifugation on Renografin-76 gradients (11) with the following modifications. Lysed cultures were lay-

Wild type

Wild type Wild type Curing Curing Conjugation Conjugation Conjugation Curing

Curing

or

reference

9 9 9 J. M. Gonzilez, Jr. (17) J. M. Gonzalez, Jr. J. M. Gonzalez, Jr.

2a 2a J. M. Gonzalez, Jr.

ered onto 32 ml of 50 to 80% linear Renografin-76 gradients prepared in 0.05% Triton X-100. The gradients were centrifuged in an SW28 rotor for 2 h at 58,000 x g. The crystal bands were collected, diluted with 3 volumes of TNT buffer, and pelleted by centrifugation in a JA-20 rotor for 20 min at 12,000 x g. The crystal fraction was washed once with TNT buffer, pelleted as described above, and resuspended in TNT buffer. The crystals were stored at 4°C. Aliquots of purified crystals were examined by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (25), and the amount of protoxin was quantified by laser densitometry. Eight serial twofold dilutions of purified crystals were prepared in 0.005% Triton X-100. Aliquots of each dilution (100 [lI) were applied to the surfaces of 30 cups (5.5-cM2 surface area) containing insect diet and dried for 1 h at 30°C. A general-purpose Noctuidae artificial diet (22) was used for Trichoplusia ni, Ostrinia nubilalis, H. virescens, and H. zea. Other diets were used for Lymantria dispar (32) and Plutella xylostella (35a). One neonate larva was added to each cup (third-instar larvae of P. xylostella), and the cups were incubated at 30°C. Mortality was recorded after 7 days. Percent toxin concentration-dependent insect mortality was transformed into probit after correction for control mortality (1). The relationship between probit and log toxin concentration can be estimated by linear regression analysis (7). Composite estimations of toxin concentrations resulting in 50% larval mortality (LC50) and 95% confidence intervals (10) were calculated from duplicated bioassays and used to compare treatments. LC50 values with nonoverlapping confidence intervals were considered significantly different. DNA sequencing. Fragments of pEG116 were cloned into M13mpl8, M13mpl9, and pTZ19U, and single-stranded templates were prepared for dideoxy sequencing with Sequenase. Oligonucleotides were synthesized on an Applied Biosystems (Foster City, Calif.) model 380A DNA synthesizer and used to progressively prime sequencing reactions. Both strands of the cry gene were sequenced. Nucleotide sequence accession number. Nucleotide sequence accession number GenBank M35524 has been assigned to the sequence of cryIA(c) from B. thuringiensis subsp. kenyae HD588-2 (see Fig. 8).

RESULTS Identification and localization of insecticidal toxin genes in B. thuringiensis subsp. kenyae. Plasmid arrays for three B.

VOL. 57, 1991

B. THURINGIENSIS SUBSP. KENYAE INSECTICIDAL TOXINS

B

A 1 -_F ~;

351

_ll

ai.

2 3 4 5 6 7 _bY NW

qw

--

VI-V

w~Ai,

i

1

2 3 4 5 6 7

_

U

130 = 115

53,51

=

44 29 -

9.6 5.4 5.2 4.9 FIG. 1. Identification of plasmid complements in B. thuringiensis subsp. kenyae and identification of native toxin-coding plasmids. Plasmid complements were visualized after modified Eckhardt lysis (A), and the gel was Southern blotted and probed with a 726-bp intragenic EcoRI fragment of crylA(a) (B). Lanes: 1, HD617; 2, HD63; 3, HD588; 4, HD1; 5, HD588-2; 6, HD73-26-45; 7, HD73-26-40. The molecular masses (in megadaltons) of the plasmid complement of HD1 are indicated. HDI contains 51- and 53-MDa plasmids that are not resolved by modified Eckhardt lysate electrophoresis (13).

thuringiensis subsp. kenyae strains are shown in Fig. 1A. The molecular masses of plasmids found in each strain were determined by comparison of their mobilities in Eckhardt lysates to the mobilities of plasmids found in B. thuringiensis subsp. kurstaki HD1 (13). B. thuringiensis subsp. kenyae HD588 (lane 3) and HD617 (lane 1) each harbor five plasmids, while HD63 (lane 2) contains nine distinct plasmids. Strain HD588-2 (lane 5) is a partially plasmid-cured derivative of HD588 that has lost the 65-MDa plasmid but retained the other four plasmids of HD588. Transconjugant strains are also shown in Fig. 1A. Strain HD73-26-45 (lane 6) has acquired the 65- and 5.3-MDa plasmids after conjugation with HD588, and strain HD73-26-40 (lane 7) has acquired the 61-MDa plasmid after conjugation with HD617. Size determinations for native plasmids present in all of these strains are summarized in Table 1. For determination of the minimal numbers and locations of insecticidal cry genes present in these strains, DNA from the gel shown in Fig. 1A was Southern transferred and probed with the 726-bp EcoRI fragment derived from the N terminus of cryIA(a). This probe is highly homologous to each subtype of cryIA (24). Strains HD588 and HD617 each contain two hybridizing plasmids (Fig. 1B). Hybridizing genes were identified on the 65- and 125-MDa plasmids of HD588 and on the 61- and 115-MDa plasmids of HD617. A single Cry-encoding plasmid (125 MDa) was found in HD63. The cured strain, HD588-2, and both transconjugant strains showed hybridization patterns consistent with these interpretations. The weaker hybridization of the large Cry-encoding plasmids probably reflects significantly fewer target sequences homologous to the probe than in the more abundant (40- to 65-MDa) mid-sized Cry-encoding plasmids. Additionally, the weaker hybridization of the large Cryencoding plasmids (115 to 125 MDa) in the B. thiuringiensis

subsp. kenyae strains than of the corresponding plasmid in strain HD1 suggests only a single copy of the cry gene on the large plasmids in the former strains. Strain HD1 contains both cryIA(a) and cryIA(c) on the 115-MDa cry plasmid (24, 42). To characterize the insecticidal genes present in various strains of B. thuringiensis, we isolated genomic DNA from the strains, digested the DNA with Hindlll, resolved the genomic fragments on an agarose gel, and Southern probed the digests with the 726-bp EcoRI fragment derived from the N terminus of cryIA(a). We found characteristic HindlIl fragments of 4.5, 5.3, and 6.6 kb associated with cryIA(a), cryIA(b), and cryIA(c), respectively, and previously described for B. thuringiensis subsp. kurstaki HD1 (24) (Fig. 2). B. thuringiensis subsp. kenyae HD588, HD617 and HD63 showed a unique hybridizing HindIII fragment of 4.8 kb that appeared to correspond to an additional type of cryIA gene. Cloning of cryIA genes from strains of B. thuringiensis subsp. kenyae. To characterize the cryIA genes that corresponded to the 4.8-kb hybridizing HindIll fragments, we cloned the cryIA genes from B. thuringiensis subsp. kenyae HD63 and derivative strains of B. thuringiensis subsp. kenyae HD588 and HD617 (Fig. 3). The toxin gene from HD63 was recovered on a 6.1-kb HpaI fragment by insertion at the unique HpaI site of pEG7. The resulting plasmid, pEG93, also contained a 1.4-kb HpaI insert fragment 3' to the fragment containing the toxin gene. The toxin gene from HD588-2 was isolated on a 9.4-kb SphI-BamHI fragment in SphI- and BamHI-digested pBR322. This plasmid was designated pEG116. We isolated the cryIA gene from the transconjugant strain HD73-26-45 on a 9.2-kb SphI-BgIII fragment that was ligated to SphI- and BamHI-digested pBR322. The resulting plasmid was named pEG118. Recovery and differentiation of clones that included each cryIA

APPL. ENVIRON. MICROBIOL.

VON TERSCH ET AL.

352

12 34 56 7 -

23.1 9.4

-

6.6

-

4.4

a

e -

2.0

Hybridization patterns associated with cryIA genes from thuringiensis subsp. kenyae. Genomic DNA was digested with HindIll, electrophoresed on a 0.7% agarose gel, Southern transferred, and probed with the 726-bp cryIA probe. The mobilities of HindlIl-digested lambda DNA fragments used as size markers are indicated (in kilobase pairs). Lanes: 1, HD588; 2, HD617; 3, HD588-2; 4, HD1; 5, HD63; 6, HD73-26-40; 7, HD73-26-45. FIG. 2.

B.

gene present in HD588 were assured by cloning toxin genes from each of these derivative strains. The cryIA gene from the 61-MDa toxin plasmid of HD617 was cloned on a

partially digested Sau3A fragment from the transconjugant strain HD73-26-40 by insertion into BamHI-digested pBR322. This plasmid was designated pEG141. The locations of the toxin genes in each of the original clones were inferred from Southern analysis (data not shown) and restriction mapping by comparison with restriction patterns predicted by the DNA sequences of cryIA(a) (35), cryIA(b) (40), and cryIA(c) (2). The mapping results strongly suggested that all four B. thuringiensis subsp. kenyae crylA genes are highly related to the cryIA(c) gene from B. thuringiensis subsp. kurstaki. To facilitate the introduction of cry genes into Bacillus

hosts,

we

constructed shuttle plasmids incorporating the

stable Bacillus replicon pBC16 (3). A 3.0-kb EcoRI fragment of pBC16 was blunt ended with the Klenow fragment of E. coli DNA polymerase and ligated to SspI-digested pUC18 (43). The resulting plasmids, pEG146 and pEG147 (Fig. 4), differ with respect to the orientation of the Bacillus replicon. Both vectors encode resistance to ampicillin and tetracycline (5 ,ug/ml) in E. coli and preserve the Lac complementation system of pUC18 for cloning in E. coli. Plasmids pEG146 and pEG147 and derivatives can be introduced and maintained in Bacillus hosts by selection for tetracycline resistance. Segregational stability studies with pEG147 and derivatives of pEG147 with cloned cryIA genes showed that these plasmids are significantly more stable in Bacillus hosts than are shuttle vector constructs that contain pBR322

sequences (results not shown). Plasmids that permitted each of the B. thuringiensis subsp. kenyae cryIA genes to be introduced into Bacillus hosts were constructed (Fig. 5). The toxin gene from pEG141 was subcloned on a 7.8-kb SphI-ScaI fragment into SphI- and

SmaI-cut pEG147. This plasmid was designated pEG149. An 11.9-kb BamHI-SphI fragment encoding cryIA from pEG116 was cloned into BamHI- and SphI-cut pNN101 (31) and transformed into B. megaterium. The resulting plasmid, pEG130, replicates only in Bacillus hosts and encodes resistance to tetracycline and chloramphenicol. A derivative of pEG118 capable of replication in Bacilllus hosts was constructed by insertion of SphI-digested pNN101 at the unique SphI site in pEG118. The resulting plasmid was designated pEG132. Several cryIA(c) genes were isolated from B. thuringiensis subsp. kurstaki strains for comparative studies with B. thuringiensis subsp. kenyae cryIA(c) genes (Fig. 6). A crylA (c) gene from B. thuringiensis subsp. kurstaki HD263-6 was recovered as a 5.5-kb Sau3A partial digestion fragment in the BainHI site of pBR322. This plasmid was designated pEG87. The crylA(c) gene from pEG87 was subcloned on a 5.4-kb SphI-AatII fragment into SphI- and AatII-digested pEG147. The resulting plasmid was designated pEG157. A cryIA(c) gene from HD73-26-10 was cloned into pBR322 on a 10.6-kb Sau3A fragment (pEG15) and then subcloned into SphI- and Stul-digested pNN101 on a 7.4-kb SphI-NruI fragment. This plasmid, pEG23, like pEG130, does not replicate in E. coli. HD73-26-10 is a transconjugant strain that carries the native 44-MDa cry plasmid of HD263. The cryIA(c) gene from B. thuringiensis subsp. kurstaki EG2069 (a derivative of EG2068 cured of the 69-MDa cry plasmid) was cloned on a 9.1-kb SphI-BgIII fragment into pBR322, generating pEG117. SphIdigested pNN101 was inserted at the SphI site in pEG117 to generate the shuttle plasmid pEG129. pEG117 and pEG129 contain the crylA(c) gene present on the 110-MDa cry plasmid of EG2069. Expression of crylA(c) genes in B. megaterium. Plasmids pEG93, pEG130, pEG132, and pEG149, containing distinct cryIA(c) genes originally from B. thuringiensis subsp. kenyae strains, and plasmids pEG23, pEG129, and pEG157, containing distinct cryIA(c) genes originally from B. thuringiensis subsp. kurstaki strains, were introduced into B. inegaterium VT1660 by protoplast transformation (39). Sporulated cultures of strains with each of these plasmids contained mixtures of bipyramidal and irregularly shaped crystalline inclusions. Crystals were purified from each of the B. megaterium strains, and equal amounts were solubilized and examined by SDS-polyacrylamide gel electrophoresis (Fig. 7). All recombinant B. megaterium strains contained a prominent protein band that migrated with a molecular mass of approximately 135 kDa, as expected for the CryIA(c) protoxin. SDS-polyacrylamide gel electrophoresis immunoblot (Western blot) results (lane 9) indicated that antibodies directed against gel-purified 130-kDa cryIA(b) toxin crossreacted with smaller proteins evident after SDS-polyacrylamide gel electrophoresis of purified crystals. This result indicated that protein bands smaller than 135 kDa in lanes 2 through 8 were probably derived from proteolysis of the 135-kDa protoxin. The amounts of CryIA(c) protein produced in recombinant B. megaterium strains were comparable, although plasmids that lacked pBR322 sequences generally produced somewhat higher (up to twofold) levels of Cry protein. This result may have been due to the increased segregational stability of pBC16-derived plasmids, which do not contain pBR322 sequences (39). The amount of Cry protein in VT1660(pEG93) was about four to five times higher than that in the other strains. Recently, others from our laboratory have shown that insertions of endotoxin genes immediately 3' to the tet gene of pBC16 are transcribed from the tet promoter during vegetative growth in B.

VOL. 57, 1991

B. THURINGIENSIS SUBSP. KENYAE INSECTICIDAL TOXINS

'0

I

--m-

i

-

L.

CL a.

I I

II

I

g% %-DAo

2L

-0

.

I

I

I

x 0.

a I

Wm

I

a

I

r-

r-

w

x

pEG116 16.0 kb

0

I

a a

u

crylA(c)

Ap

HD 588-2 ?I 'i CC8

-

I

I

I

I

II

0

2

U)

I

I

I

xw I

I

CC

-

-

-

E-0

-0-

W8 a. a.UNx

_~

__

(A

I

II

I

I

I CL

CL I

I

I

wc XI



a

c

pEG141 17.5 kb

~~~~~~~crylA(c)

I

Ap

< HD 617 Iww~~s cp a.-a. CL gCco ;( tiXlX AIct IIC 63an crylA(c)

I FB

353

0.L

I 31

a.1 C

pEG93 17.5 kb

1.0 kb FIG. 3. Restriction maps for primary isolations of crylA(c) from B. thuringiensis subsp. kenyae HD588, HD617, and HD63. The localization and orientation of cry genes were determined by restriction and Southern mapping. Symbols: vector sequences derived from vector sequences derived from pNN101. pBR322; Lii,

,

lated cultures had numerous sporangial mother cells that contained large crystals but no spores. These effects were also observed with B. megaterium strains that express cryIIIA (8). Similar, although less pronounced, effects were seen for cloned cryIA(c) genes in B. thuringiensis hosts (37b). Insecticidal activities of CryIA(c) proteins from B. thuringiensis subsp. kenyae and B. thuringiensis subsp. kurstaki.

megaterium and B. thuringiensis (29). Insertions within the tet structural gene (as in pEG93) are probably also expressed during vegetative growth and may serve as the basis for the increased accumulation of toxin in VT1660(pEG93). The expression of all crylA(c) genes in B. megaterium resulted in reductions in spore titers of about 1 order of magnitude, as compared with the spore titers in control strains containing the expression vectors. In addition, sporu-

w

I

I

1.

pEG146 5.8 kb

I I

I I

Ap MCS

TC la

cc

8

M=

CL

7. .g

A .%

11 I

I

U 8

w

H

A

.

I

if

-

I

w

i_cl

3 __ _

Cxxxx,sa

2

A, MCS

Ap

E

1

pEG147 5.8 kb

TC

1.0 kb

sequences FIG. 4. Restriction maps of shuttle vector plasmids pEG146 and pEG147. Symbols: =II, sequences derived from pUC18; derived from pBC16. MCS, Multiple cloning site of pUC18 containing unique sites for Hindlll, SphI, PstI, Sall, XbaI, BamHI, SmaI, KpnI, Sacl, and EcoRI. All of these sites are also unique in pEG146 and pEG147. ,

354

APPL. ENVIRON. MICROBIOL.

VON TERSCH ET AL.

m

_o

-&

_

a-. a-.

-

-

l n'

-

L

E

cox:

cL I I

Ap

&

III

1

-

I

3

I

I

-LX mg -2J I

I

crylA(c) HD 588

Tc

On

#A 1

1

1

1

1

1

2

I

l

p

wtr~r'4

18.9 kb

i iI I mg 1 K

. a-.

I

-

I=

pEG130

I1

II

17.4 kb

crylA(c) HD 5882

Tc

On

co

E

_~I

_ I

l1

_

=S

A

_

C,,_

l__L _ X _i

I

I I I

I

I

I

l

.... .... .. . ..

crylA(c)

1.0 kb

pEG149 13.9 kb

. . III III ~ W

%_

HD 617 FIG. 5. Restriction maps of clones of cryIA(c) from B. thiuringiensis subsp. kenyae HD588, HD617, and HD63 capable of replication in Bacillus hosts. Symbols: sequences derived from pBR322; sequences derived from Bacillus plasmid pNN101; sequences derived from shuttle plasmid pEG147. LII,

,

,

Purified crystals from recombinant B. megaterium strains were assayed quantitatively against larvae of six pest lepidopteran insects. CryIA(c) crystal proteins of each subspecies exhibited similar specific insecticidal activities against some lepidopteran insects (Table 2). Small differences in specific activities were observed for all seven Cry proteins when assayed against H. zea or L. dispar. Activities against T. ni were also about the same for B. thuringiensis subsp. kenyae and B. thuringiensis subsp. kurstaki toxins, although the latter encoded by pEG129 may be somewhat less active than those encoded by the other B. thuringiensis subsp. kurstaki cryIA(c) genes. The B. thuringiensis subsp. kenyae CryIA(c) proteins tended to be somewhat less active against H. virescens than did the B. thuringiensis subsp. kurstaki

CryIA(c) proteins. These differences are probably not significant, for the 95% confidence intervals for LC50 estimates for the toxins of each subspecies showed considerable overlap. In contrast, each of four B. thuringiensis subsp. kenyae CryIA(c) toxins was significantly less active against P. xylostella than was each of three B. thuringiensis subsp. kurstaki CryIA(c) toxins. In these assays, clear separation of the 95% confidence intervals was observed in all pairwise comparisons of both toxins. B. thuringiensis subsp. kenyae CryIA(c) toxins appeared less active than did B. thuringiensis subsp. kurstaki CryIA(c) toxins against 0. nubilalis, although the separation of confidence intervals in pairwise comparisons was not as pronounced or as consistently observed as in assays of P. xylostella.

z

z L _ v_ A__ _

L

r~~~~~~~~~I I:_ ul,nju

IL0

-,

pEGZ3 12.6 kb

DotS

IC

T.-

HD23

m

SE

o

aw-~~~

I =0.L I

i

I

-

I I I

Ill

I

Il

,,,,,

I

.

*

Ap

Tc

l

'

gpEG1

18.8 kb

c

Orn